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DNA, RNA, Protein Sample Prep & DNA Amplification
2000, Dr. Alka Agrawal Transposition and evolution of antigen-specific immunity Dr. Agrawal earned her bachelor's degree in chemical engineering from the University of Michigan.
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Having entered the pharmacology graduate school program at Yale University, she began working in the laboratory of David Schatz investigating the role of DNA repair proteins in V(D)J recombination. The development of an in vitro V(D)J cleavage system in the laboratory changed her focus and she began studying the cleavage mechanism as part of her doctoral research. Exposure to science policy at the National Academy of Sciences, and to science journalism through the AAAS Mass Media Science and Engineering Fellows Program, prompted Dr. Agrawal to pursue a career in science writing.
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GE & Science Prize for Young Life Scientists is supported by GE Healthcare and the journal Science, which is published by AAAS, the nonprofit society. Used with permission of AAAS © 2007. Further information on how to enter, plus past winners and their essays can be found at:
www.gelifesciences.com/science
ch05.fm Page 146 Tuesday, June 17, 2008 11:47 PM
Introduction Introducing illustra and Trap for Nucleic Acid and Protein Sample Preparation
J Preparing protein, DNA, and RNA samples is just as critical as the analysis step. J Reducing errors and increasing reproducibility at the preparation stage ensures the best results. J Genomic and protein research workflows involve combinations of biomolecules and are becoming more complex. J Identifying the right product for each stage of the research process is important for obtaining the best results.
J Straightforward protocols to generate reproducible results with exceptional yield and purity. J Simplicity through reliable kit design and ready-to-use components. J Optimized protocols for specific proteins and analytical techniques such as electrophoresis, liquid chromatography, and mass spectrometry. The products are packed in appropriate formats for typical sample volumes analyzed using these techniques and and are packed in quantities for small-series and for high-throughput requirements. Regardless of your sample source, purification target, or downstream application, these products enable you to get a great start for an excellent finish.
We have simplified the process and made it easier for users to get started by providing the right product for each stage of the process.
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RTG PCR kits
DNA/RNA isolation kits
TempliPhi/GenomiPhi kits PAGE: 146 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
The illustra platform for nucleic acid preparation and the Trap platform for protein preparation provide high standards of quality, giving you:
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A good start leads to a great finish
GE Healthcare understands the challenges that need to be met in life science research. For over 50 years we have been developing analytical techniques and biomolecule preparation chemistry. Today, we are continuously working with our customers to optimize their entire workflow. That is why our sample preparation products have been designed with these principles in mind:
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5
Get it right from the start
MultiTrap 96-well plates for protein enrichment, capture, cleanup, and expression screening
SpinTrap and Buffer kits for protein enrichment, antibody purification, and sample cleanup
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 147 Tuesday, June 17, 2008 11:47 PM
Introduction WORKFLOW – Nucleic Acid Amplification and Purification
5
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DNA, RNA, Protein Sample Prep & DNA Amplification
GE Healthcare
For more information, visit www.gelifesciences.com
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Introduction
Genomic DNA Preparation
Paraffin-embedded tissue or difficult samples Cultured cells
DNA, RNA, Protein Sample Prep & DNA Amplification
5
148
Blood
Buffy coat
Nucleated red blood cells
Bone marrow (suspended cells) Bacteria (Gram - and +)
Plants
FTA paper/Guthrie card Buccal swab
Purified genomic DNA
Downstream applications
Genotyping Cloning & sequencing
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> 300 cells (4 to 7 µg DNA yield) > 300 cells (40 to 50 µg DNA yield) 5 to 50 mg Up to 200 mg Up to 25 mg 30 m paraffin section > 300 cells (4 to 7 µg DNA yield) > 300 cells (40 to 50 µg DNA yield) Up to 5.0 x 105 cells 1 to 3 x 105 Up to 2.0 x 107 cells 3 x 106 to 1 x 107 cells 5 to 10 µl (4 to 7 µg DNA yield) 5 to 10 µl (40 to 50 µg DNA yield) 50 to 300 µl 1 ml 1 to 8 ml 10 ml 5 to 10 µl (4 to 7 µg DNA yield) 5 to 10 µl (40 to 50 µg DNA yield) 50 to 300 µl 10 µl (4 to 7 µg DNA yield) 10 µl (40 to 50 µg DNA yield) 10 µl 25 to 200 µl (tested) 5 to 10 µl (4 to 7 µg DNA yield) 5 to 10 µl (40 to 50 µg DNA yield) 200 µl > 300 cells (4 to 7 µg DNA yield) > 300 cells (40 to 50 µg DNA yield) Up to 4.0 x 109 1 cm leaf (4 to 7 µg DNA yield) 1 cm leaf (40 to 50 µg DNA yield) 1 seed (4 to 7 µg DNA yield) 1 seed (40 to 50 µg DNA yield) Up to 1.0 g 3 x 3 mm piece (4 to 7 µg DNA yield) 3 x 3 mm piece (40 to 50 µg DNA yield) Single swab Single swab (4 to 7 µg DNA yield) Single swab (40 to 50 µg DNA yield) > 10 ng (4 to 7 µg DNA yield) > 10 ng (40 to 50 µg DNA yield)
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Animal tissue
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Starting quantity (DNA yield)
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Sample types
tis su e tis & c su ell sM e i bl & c oo el ni S ls pi d n M M ( i bl oo ini S di F see lo pi d pa w n M ba g ct idi F (see (see e 1 er lo pa pa 53) i w a BA g g CC Min (see e 1 e 1 iS 55 54 1 p ) ) BA (se pin ag CC e p (s e ag ee 156 2 pa ) Nu & e g cle BAC 15 9) e 1 o C 57 Ph n 3 (s ) yt HT op (s ee e p Ge ure e a pa ge no ( g 1 m se Ge iP e p e 15 59) no hi V ag 9 ) e m 2 1 ( iP hi see 59) HY pa (se ge 1 e pa 60 ge ) 16 1)
SELECTION GUIDE – Genomic DNA
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For more information, visit www.gelifesciences.com
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GE Healthcare
ch05.fm Page 149 Tuesday, June 17, 2008 11:47 PM
Introduction
Ex oS A GF P-I X T (s PC e GF R a e p a X 9 nd ge M 6 P Ge 18 icr CR l B 3 o a ) Au Spi (see nd (se to n G p a S e p Pr eq -25 ge ob G- (s 18 ag eQ 50 ee 2) e 1 81 NI p CK uan (see ag ) co t G pa e 18 NA lu -5 g 6 P- mn 0 (s e 1 ) 84 NA 5 co s (s ee ) P- lum ee pag 1 p NA 0 c ns ag e 18 P- olu (se e 1 5) 8 m e 2 Cy 5 c ns pa 5) Sc olu (se ge r m 1 n ep 8 M ibe icr G s (s ag 6) oS FX ee e 1 M pin (se pa 86 icr ) e o Gp ge M Spin 50 age 186 i cr ( s oS S-2 ee 18 ) M pin 00 pa 5) i cr g oS S-3 (see e 1 00 p 84 pi a n ( S- se ge ) 40 e p 18 0 (se age 4) e pa 183 ge ) 18 2)
SELECTION GUIDE – DNA Cleanup
DNA Cleanup
Sample types
Starting quantity
PCR products
Any size, for sequencing only 50 bp to 10 kb, few samples 100 bp to 10 kb, many samples 10 to 50 bp 50 kbp to 10 bp
Agarose gel slices or enzyme removal Sequencing reactions Labeled DNA fragments
A range of volumes and sample sizes
Downstream applications
• • •
12 to 25 µl > 20 bases > 10 bases 100 µg sample in 100 µl or no microcentrifuge 100 to 150 µl 0.1 to 0.5 ml 0.5 to 1 ml 1 to 2.5 ml CyDye labeled probes 25 to 50 µl for labeled DNA 10 to 100 µl for buffer exchange or desalting
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PCR (25 to 50 µl) or labeling reaction (25 to 50 µl) < 100 bp PCR (25 to 50 µl) or labeling reaction (50 to 75 µl) or fragment < 200 bp PCR (50 to 100 µl) or labeling reaction (75 to 100 µl) or remove primers > 24 bases Cloning & sequencing Gene expression Genotyping
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SELECTION GUIDE – RNA Preparation
RNA Preparation PAGE: 149 - CURRENCY: USD
5
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Sample type and format
Starting quantity
Total RNA Cultured cells and tissue Tube format 96-well plate vacuum 96-well plate centrifuge mRNA purification Eukaryotic cells or tissue Eukaryotic total RNA
Up to 200 mg tissue or 5 x 107 cells 10 to 30 mg or up to 2 x 106 cells 10 to 30 mg or up to 2 x 106 cells 30 mg or up to 1 x 107 cells 1 to 5 x 107 cells or 0.5 g tissue 1 to 1 x 107 cells or 100 mg tissue Total RNA or 25 mg to 1 g tissue
Downstream applications
Gene expression
GE Healthcare
RN As p RN in M As in i p RN in M (see pa As i p di g Qu in 9 (see e 1 pa 68 ick 6 ( ) s Qu Pre ee ge 1 ick p m pag 69 ) P e R 17 m rep NA RN M (se 0) i A Pu cro e p m a rif ica RN ge tio A ( 171 s n ) Ki ee p t( se ag e e pa 17 ge 1) 17 2)
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cDNA A range of sample types PCR, sequencing, and labeling reactions
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DNA, RNA, Protein Sample Prep & DNA Amplification
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Oligonucleotides
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Introduction
Ho tS Ho tart t S Mi x t Pu art RT Re M G* a RT Taq ste (see G* R r M p T a RT RT- G* P ix (s ge -P PC C ee 17 R R C p 3 Fid R M (se (se ag ) el as e p e p e 1 iT a a 7 t Fid aq er M ge ge 4) el PC ix 17 17 iTa R 5) 8 M (see ) Fid q el PC ast pa er iTa R g Fid q M M e el RT ast ix 176 iTa -P er (2 ) Ta q C M x) (s i D R q DN NA Ma x (P ee lu pa A P s Po oly ter s) (s ge lym m Mix ee 17 e er ras (2x pa 6) as e ) (s ge ( e (C see ee 176 lo ne pag pag ) d) e e (se 18 179 0 e ) pa ) ge 17 9)
SELECTION GUIDE – PCR & RT-PCR
PCR and RT-PCR
Starting quantity
PCR
Basic PCR (amplicon up to 3 kb) Hot start PCR (amplicon up to 3 kb) Long range PCR (amplicon up to 20 kb) Basic RT-PCR (amplicon up to 3 kb) Long range RT-PCR (amplicon up to 6 kb)
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Genotyping Cloning & sequencing Gene expression
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RT-PCR
Downstream applications
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*RTG is an abbreviation of Ready-To-Go, a single-dose, room-temperature-stable, bead format that contains all PCR or RT-PCR components. You just need to add DNA template, primer, and water for a reaction.
Starting quantity
Bacterial culture
1 µl (high throughput) 1 µl (low to medium throughput) 1 to 3 ml 25 to 500 ml < 1 colony (high throughput) < 1 colony (low to medium throughput) < 1 µl (high throughput) < 1 µl (low to medium throughput) > 1 ng (high throughput) > 1 ng (low to medium throughput) > 1 ng
Bacterial colony Bacteria glycerol stock Purified DNA (small vectors) Purified BAC/fosmid DNA Difficult templates (GC rich, secondary structures) M13 phage plaque M13 phage glycerol stock Downstream applications
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0.1 to 1 ng < 1 plaque < 1 µl Cloning Sequencing
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*Can be used directly for PCR and subcloning. Addtional steps will be needed for transformation or transfection
150
For more information, visit www.gelifesciences.com
GE Healthcare
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Sample types
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Plasmid DNA Preparation
pl as m pl idPr as ep m M Te idPr ini m (s ep pl M ee p iP i Te h d ag m i Se i (s e pl qu ee 1 i P Te en pa 62) h m i1 g c e e pl 0 Te iPh 0/5 Tem 163 m i L 00 pl ) a pl iP rge * (se ate hi Se Co e pa Pre p qu nst g en ru e 1 Kit* ce ct* 65 (se Re (se ) e pa so e ge lve pa g 16 rK e 5) it* 16 (se 7) e pa ge 16 6)
SELECTION GUIDE – Plasmid DNA
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DNA, RNA, Protein Sample Prep & DNA Amplification
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Amplification method
ch05.fm Page 151 Tuesday, June 17, 2008 11:47 PM
Introduction SELECTION GUIDE – Trap Protein Sample Preparation Products
See page
2-
Code No.
D
Protein Sample Preparation
1D
el ec tro ph el Liq ectr ore s o ui ph is d Fr ch ore om ro s m is Fr 2-D ato om e le gra Fr 1-D ctro phy om e ph le o Pr LC ctro res ot to ph is t ei M M n e S ore o M as xp sis S s s re to pe s s M ct ion S ro m et ry
Choose analysis method
Untagged Protein Enrichment •
28-9135-68 pg. 197 28-9031-31 pg. 198 28-9031-32 pg. 199
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28-9031-33 pg. 200 28-9031-34 pg. 201
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Ab SpinTrap
28-9135-67 pg. 201 28-9031-35 pg. 202 28-4083-47 pg. 467
Immunoprecipitation Starter Pack Phos SpinTrap Fe
17-6002-35 pg. 203 28-9298-81 pg. 204
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Streptavidin HP SpinTrap Streptavidin HP SpinTrap Buffer Kit Streptavidin HP MultiTrap Protein A HP SpinTrap Protein A HP MultiTrap Protein G HP SpinTrap
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Protein A/G HP SpinTrap Buffer Kit Protein G HP MultiTrap
• Bulk NHS HP Sepharose™ and empty spin columns for coupling of an affinity molecule with a primary amine • Buffers for immunoaffinity enrichment using NHS HP SpinTrap • Coupling of a biotinylated affinity molecule, spin column format
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• Buffers for immunoaffinity enrichment using Streptavidin HP SpinTrap • Coupling of a biotinylated affinity molecule, larger sample series or automation • Coupling of an IgG affinity molecule, spin column format
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• Coupling of an IgG affinity molecule, large sample series, or automation • Coupling of an IgG affinity molecule, spin column format • Buffers for immunoaffinity enrichment using Protein A HP SpinTrap, Protein G HP SpinTrap, and Ab SpinTrap
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• Coupling of an IgG affinity molecule, larger sample series, or automation • Coupling of an IgG affinity molecule, spin column format, larger pack size • Coupling of an IgG affinity molecule, batch application • Enrichment of phosphorylated peptides, spin column format, IMAC precharged with Fe
Small-scale Antibody Purification Protein A HP SpinTrap Protein A HP MultiTrap Protein G HP SpinTrap Protein G HP MultiTrap Ab SpinTrap Ab Buffer Kit
28-9031-32 pg. 199 28-9031-33 pg. 198 28-9031-34 pg. 201 28-9031-35 pg. 202
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Purification of IgG, spin column format
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Purification of IgG, spin column format
28-4083-47 pg. 467 28-9030-59 pg. 468
• •
Purification of IgG, spin column format, larger pack size
Purification of IgG, larger sample series, or automation
Purification of IgG, larger sample series, or automation
Buffers for antibody purification using Protein A HP SpinTrap, Protein G HP SpinTrap, and Ab SpinTrap
Desalting/Buffer Exchange/Cleanup
PD SpinTrap G-25 PD MultiTrap G-25
28-9180-04 pg. 205 28-9180-06 pg. 206
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• Clean-up of proteins/oligosaccharides by gravity flow and spin flow, sample volumes up to 2.5 ml • Buffer reservoir for easy equilibration • Clean-up of proteins/oligosaccharides by gravity flow and spin flow, sample volumes up to 1.0 ml • Clean-up of proteins/oligosaccharides by gravity flow and spin flow, sample volumes up to 0.5 ml • Clean-up of proteins/oligosaccharides by microcentrifugation, sample volumes up to 130 µl • Clean-up of proteins/oligosaccharides in parallel, 96-well filter plates by vacuum or centrifuge,
PD MidiTrap G-10 PD MiniTrap G-10 Mini Dialysis Kit - 1 kDa cut-off Mini Dialysis Kit - 8 kDa cut-off 2D Clean-Up Kit SDS-PAGE Clean-Up Kit
28-9180-11 28-9180-10 80-6483-75 80-6484-13 80-6484-51 80-6484-70
pg. 210 pg. 209 pg. 211 pg. 211
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pg. 195 pg. 212
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80-6501-23 pg. 191 80-6501-42 pg. 191
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80-6501-04 pg. 194
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80-6483-56 pg. 213
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pg. 192
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80-6483-37 pg. 191
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Disposable PD-10 Desalting columns 17-0851-01 pg. 503 LabMate PD-10 Buffer reservoir 18-3216-03 pg. 503 PD MidiTrap G-25 28-9180-08 pg. 208 PD MiniTrap G-25 28-9180-07 pg. 207
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sample volumes up to 130 µl, large sample series and automation Peptide and small protein applications, clean-up of sample volumes up to 800 µl by gravity flow Peptide and small protein applications, clean-up of sample volumes up to 300 µl by gravity flow Dialysis of small sample volumes, 250 µl and 2 ml Dialysis of small sample volumes, 250 µl and 2 ml
• Removal of interfering contaminants and concentration of total protein • Removal of interfering contaminants
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Enzyme Regulation Protease Inhibitor Mix Nuclease Mix
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• Protease inhibitor cocktail • Cocktail of nucleases for the removal of DNA and RNA from the protein sample
Fractionation 2D Fractionation Kit
• Fractionation of the total protein in sample in six discrete fractions
Total Protein Quantitation 2D Quant Kit
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• Quantitation of the protein amount using a colorimetric assay
Protein Depletion Albumin and IgG Removal Kit
RPN6300
Removal of albumin and IgG from a protein sample, spin format
Lysis/Tissue Homogenization Sample Grinding Kit
GE Healthcare
•
• The mechanical breaking up of cells or tissue releasing the total protein content
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
28-9031-28 pg. 196 28-9135-69 pg. 196 28-9031-30 pg. 197
NHS HP SpinTrap NHS HP SpinTrap Buffer Kit
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Introduction SELECTION GUIDE – Trap Protein Sample Preparation Products
See page
1D
Code No.
2D
el
Protein Sample Preparation
ec tro ph el e Liq ctr ore s o ui ph is d Fr ch ore om ro s m is Fr 2-D ato om e g le ra Fr 1-D ctro phy om e ph le o Pr LC ctro res ot to i ei M pho s to n S M re as exp sis MS s s re to pe ss M ct ion S ro m et ry
Choose analysis method
Histidine-tagged Protein Capture His GraviTrap His GraviTrap Kit His MultiTrap HP
His SpinTrap Kit His Buffer Kit
Gravity flow columns for histidine tagged protein purification and buffers
28-4009-90 pg. 426 28-4013-53 pg. 420 28-9321-71 pg. 420 11-0034-00 pg. 429
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Purification of histidine-tagged proteins in larger series or automation in 96-well format
• •
Purification of histidine-tagged proteins in larger series or automation in 96-well format
• •
Microspin columns for histidine-tagged protein purification and buffers
Purification of histidine-tagged proteins, spin column format
Buffers for the purification of histidine-tagged proteins using His GraviTrap, HisTrap, or His SpinTrap
• •
Unconjugated antibody with affinity for the histidine tag
HisTrap FF Columns
27-4710-01 pg. 429 17-5247-01 pg. 418 17-5319-01 pg. 422
•
Simple purification with a syringe or chromatography system such as ÄKTAdesign
HisPrep FF 16/10 Column HisTrap FF Crude Kit
17-5256-01 pg. 425 28-4014-77 pg. 424
• •
Easy scale-up purification of histidine-tagged proteins
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Purification of GST-tagged proteins in larger series or automation in 96-well format
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Purification of GST-tagged proteins in larger series or automation in 96-well format
• •
Purification of GST-tagged proteins, spin column format
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Sequencing primers for pGEX vectors
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Bacterial strain for the expression of GST fusion protein
Bulk GST Purification Module PreScission Protease
28-4055-01 pg. 443 28-4055-00 pg. 443 27-4570-03 pg. 442 pg. 431 multiple pg. 433 multiple 27-1542-01 pg. 451 27-1524-01 pg. 450 27-4570-01 pg. 442 27-0843-01 pg. 445
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Bulk Glutathione Sepharose 4B and empty gravity columns
Thrombin Protease
27-0846-01 pg. 444
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Purified enzyme for site-specific cleavage of the GST tag fr om fusion proteins expressed using the pGEX-T vectors
Factor Xa Protease
27-0849-01 pg. 444
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Purified enzyme for site-specific cleavage of the GST tag fr om fusion proteins expressed using the pGEX-X vectors
Anti-GST Antibody GST Detection Module
27-4577-01 pg. 446 27-4590-01 pg. 445
• •
Unconjugated antibody with affinity for the GST tag
GST 96-well Detection Module
27-4592-01 pg. 446 17-5234-01 pg. 440 17-5281-01 pg. 437 17-5130-02 pg. 439 28-4017-45 pg. 441 27-4570-03 pg. 442
Anti-His Antibody HisTrap HP Columns
Simple purification with a syringe or chromatography system such as ÄKTAdesign
Purification with a syringe or chromatography system e.g., ÄKTAdesign, no filtration of the sample
GST-tagged Protein Capture GST MultiTrap FF GST MultiTrap 4B GST SpinTrap Purification Module pGEX Vectors GST Vector Primers for Sequencing E. coli BL21 M13K07 Helper Phage
GSTrap FF Columns GSTrap 4B Columns RediPack GST Purification Module
M13 phage for the GST expression system
Purified enzyme for site-specific cleavage of the GST tag fr om fusion proteins expressed using the pGEX-P vectors
Reagents for the biochemical reaction performed by the GST-tag for the determination of concentration
•
Reagents and plates with precoated wells for the detection of GST-tagged proteins
• •
Simple purification with a syringe or chromatography system such as ÄKTAdesign
•
Simple purification with a syringe or chromatography system such as ÄKTAdesign
•
Simple purification with a syringe or chromatography system such as ÄKTAdesign
•
Purification of GST-tagged proteins in gravity-flow format
28-9187-78 pg. 447
•
Simple purification with a syringe or chromatography system such as ÄKTAdesign
28-9075-46 pg. 448
•
Simple purification with a syringe or chromatography system such as ÄKTAdesign
Simple purification with a syringe or chromatography system such as ÄKTAdesign
Maltose Binding Protein (MBP)-tagged Protein Capture MBPTrap HP Strep(II)-tagged Protein Capture StrepTrap HP Sample Concentration Vivaspin
multiple
pg. 193
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Concentration of up to 20 ml sample through filter-based ultracentrifugation
For more information, visit www.gelifesciences.com
GE Healthcare
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GSTPrep FF 16/10 Column GSTrap HP Columns
Expression vectors for GST fusion proteins
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152
Purification of histidine-tagged proteins, gravity flow column
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DNA, RNA, Protein Sample Prep & DNA Amplification
5
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His MultiTrap FF His SpinTrap
11-0033-99 pg. 428 28-4013-51 pg. 428 28-4009-89 pg. 426
ch05.fm Page 153 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Genomic DNA Preparation illustra tissue and cells genomicPrep Mini Spin Kit
PAGE: 153 - CURRENCY: USD
The kit contains spin columns prepacked with a novel silicamembrane, lyophilized proteinase K powder, lysis solutions, wash and elution buffers, 2 ml microcentrifuge collection tubes and a full protocol booklet with a detachable, quick reference protocol card. References 1. Human Genome Resources [Online] http://www.ncbi.nlm.nih.gov/genome/guide/human 2. Sambrook, J. and Russell, D. W. Molecular Cloning, A Laboratory Manual, chapter 6 (2001). 3. Vogelstein, B. and Gillespie, D. Proc. Natl. Acad. Sci. USA 76, 615 (1979).
Product
50
28-9042-75
illustra tissue and cells genomicPrep Mini Spin Kit
250
28-9042-76
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Master Mix illustra Hot Start Mix RTG
page 174 page 173
illustra tissue and cells genomicPrep Midi Flow Kit RT-PCR Master Mix (2X)
page 154 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs
page 188
ExoSAP-IT illustra GenomiPhi HY DNA Amplification Kit
page 183 page 161
illustra GenomiPhi V2 DNA Amplification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 160 page 181
illustra GFX 96 PCR Purification Kit illustra AutoSeq G-50
page 182 page 184
TECHNICAL SPECIFICATIONS Tissue & cells Mini Spin (5 to 50 mg of Rat Liver; 20 mg optimum) Yield Purity Total time of prep* Hands-on time* Size
12.0 -25.0 µg 1.75 to 1.90 90 min 20 min 20 to 35 kb
*prep time does not include homogenization
Tissue & cells Mini Spin ( 3 u 105 - 5 u 106 cells) Yield Purity Total time of prep* Hands-on time* Size
13 to 17 µg 1.75 to 1.90 70 min 20 min up to 75 kb
* Cell type dependent, CHO and HK293 cells used in testing
Sizing of genomic DNA using pulse-field gel (PFG) electrophoresis. Genomic DNA (200 ng) isolated with the illustra tissue and cells genomicPrep Mini Spin Kit by three different researchers were ran in lanes 3 to 11. Genomic DNA (200 ng) purified with the QIAamp DNA Mini Kit (Qiagen) was ran in lanes 13 to 14. Control rat genomic DNA (Bioline) was ran in lane 15. Low- and high-range PFG markers (New England Biolabs) of lambda DNA embedded in agarose plugs were ran in lanes 1 and 2, respectively.
GE Healthcare
Quantity Code Number
illustra tissue and cells genomicPrep Mini Spin Kit
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To minimize the incidence of DNA shearing during extraction, the illustra tissue and cells genomicPrep Mini Spin Kit includes a lysis solution with proteinase K to gently release genomic DNA from tissues or cells. The kit can be used to extract DNA from diverse tissue types such as liver, kidney, and mouse tails with yields ranging from 0.5 to 1.5 µg of genomic DNA per mg of tissue. The kit consistently generates purified genomic DNA with optical density A260/280 readings of about 1.8. The illustra tissue and cells genomicPrep Mini Spin Kit can be used to isolate genomic DNA from 1 to 5 u 106 mammalian cells. Genomic DNA yields vary according to the cell line with 10 to 13 µg per 5 million CHO cells and about 40 µg from a human lung fibroblast cell line such as MRC5.
ORDERING INFORMATION
For more information, visit www.gelifesciences.com
HindIII digest of genomic DNA purified with the illustra tissue and cells genomicPrep Mini Spin Kit (lanes 3– 5) and the QIAamp DNA Mini Kit (Qiagen) (lane 6), respectively. Uncut DNA purified with the illustra kit was ran in lane 2 as control. LambdaHindIII ladder was ran in lane 1.
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 153 - CURRENCY: USD
J For the rapid extraction and purification of high-quality genomic DNA from a variety of animal tissues and mammalian cell cultures. J Capable of purifying up to 1.5 µg/mg of tissue from an input tissue sample range of 5 to 50 mg, and up to 40 µg of genomic DNA per 5 million cells depending on cell type. J Total DNA extraction time reduced significantly to 90 min. J Produces high-quality genomic DNA with high molecular weight and less degradation. J Validated in several downstream applications including realtime PCR, endpoint PCR, multiplex PCR, and restriction enzyme digests.
153
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Nucleic Acid Sample Preparation Genomic DNA Preparation illustra tissue and cells genomicPrep Midi Flow Kit 25
28-9042-73
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Master Mix
page 174
illustra Hot Start Mix RTG RT-PCR Master Mix (2X)
page 173 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs ExoSAP-IT
page 188 page 183
illustra tissue and cells genomicPrep Mini Spin Kit
page 153
illustra GenomiPhi HY DNA Amplification Kit illustra GenomiPhi V2 DNA Amplification Kit
page 161 page 160
illustra GFX PCR DNA and Gel Band Purification Kit illustra GFX 96 PCR Purification Kit
page 181 page 182
illustra AutoSeq G-50
page 184
TECHNICAL SPECIFICATIONS Tissue & cells Midi (50 to 200 mg of tissue; 100 mg optimum) Yield Purity Total time of prep* Hands-on time* Size
100 to 135 µg 1.7 to 1.9 3h 1h y 50 kb
*prep time does not include homogenization
Tissue & cells Midi (1 u 107 to 2 u 107; 2 u 107 optimum) Yield Purity Total time of prep* Hands-on time* Size
100 to 135 µg 1.7 to 1.9 3h 1h y 50 kb
*including lysis, purification & desalting
References 1. Aljanabi, S. M. and Martinez, I. Nucl. Acids Res 25, 4692–4693 (1997). 2. Vogelstein, B. and Gillespie, D. Proc. Natl. Acad. Sci. USA 76, 615 (1979). 3. Sambrook, J and Russell, D. W. Molecular Cloning, A Laboratory Manual , Chapter 6 (2001).
97 kb 48 kb 23 kb
1
Sizing of genomic DNA using pulse-field gel (PFG) electrophoresis shows highquality genomic DNA isolated from rat liver tissue using the tissue and cell genomicPrep Midi Flow Kit. Lanes 1 and 2 are lambda and low-range markers, respectively. Lanes 21 and 22 contain low range and lambda markers. Lanes 3 to 14 represent genomic DNA purified with the tissue and cell genomicPrep Midi Flow Kit. Lanes 15 to 20 represent genomic DNA purified with a Qiagen blood & cell culture midi kit.
2
3
4
5
6
Lanes 1, 3, and 5 represent the samples digested with EcoRI for 2 h. Lanes 2, 4, and 6 represent genomic DNA used in the digestion experiments.
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 154 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Quantity Code Number
illustra tissue and cells genomicPrep Midi Flow Kit
PAGE: 154 - CURRENCY: USD
The kit contains fast-flow genomic 250 columns, lyophilized proteinase K powder, lysis solution, elution buffers, a NAP-25 desalting column, spin column adapters, a full protocol, and a detachable, quick reference protocol card.
Product
PAGE: 154 - CURRENCY: USD
154
The kit can be used to isolate up to 250 µg of high-quality genomic DNA from an input sample of 50 to 200 mg of tissue and up to 2 u 107 cells. The genomic DNA product is suitable for most biological applications including PCR, real-time PCR, restriction enzyme digests, and sequencing. The illustra tissue and cells genomicPrep Midi Flow Kit utilizes a simple and efficient three-step process involving a one-step lysis procedure, anion-exchange column chromatography, and a highly reproducible desalting process.
ORDERING INFORMATION
PAGE: 154 - CURRENCY: USD
5
J For the high-yield extraction and purification of highquality genomic DNA from tissues and cells—20% higher yield than other commercially available kits. J High-quality genomic DNA with a greater proportion of higher molecular weight DNA and less shearing. J Increased input capacity of 200 mg of tissue or up to 2 u 107 cells and a higher resin capacity of 250 µg. J Validated in several downstream applications including realtime PCR, endpoint PCR, Multiplex PCR, and restriction enzyme digests. J Shorter total DNA extraction time of 3 to 4 h and less hands-on time due to the inclusion of NAP-25 desalting column, removing the need for time consuming and complicated isopropanol precipitation. J Flexibility to use our pre-equilibrated fast-flow resin columns in spin or gravity mode.
ch05.fm Page 155 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Genomic DNA Preparation illustra blood genomicPrep Mini Spin Kit
PAGE: 155 - CURRENCY: USD
Product
Quantity Code Number
illustra blood genomicPrep Mini Spin Kit
50
28-9042-64
illustra blood genomicPrep Mini Spin Kit
250
28-9042-65
For pricing information, visit www.gelifesciences.com/orderonline Related Products illustra Hot Start Master Mix
Refer To page 174
illustra Hot Start Mix RTG
page 173
illustra blood genomicPrep Midi Flow Kit RT-PCR Master Mix (2X)
page 156 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs ExoSAP-IT
page 188 page 183
illustra GenomiPhi HY DNA Amplification Kit
page 161
illustra GenomiPhi V2 DNA Amplification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 160 page 181
illustra GFX 96 PCR Purification Kit illustra AutoSeq G-50
page 182 page 184
TECHNICAL SPECIFICATIONS Yield Purity Total time of prep* Hands-on time* Size
4 to 6 µg/200 µl whole y 1.7 to 1.6 less than 20 min less than 5 min y 20 kbp
*including lysis, purification & desalting
References 1. Sambrook, J., Fritsch, E.F and Maniatis, T. Molecular cloning: A laboratory Manual, Cold Spring Harbor laboratory, 2nd eds., (1989). 2. Vogelstein, B. and Gillespie, D. Proc. Natl. Acad. Sci. USA 76, 615 (1979). 3. Marko, M.A., Chipperfield, R. and Birnboim, H.C. Anal. Biochem. 121, 382 (1982). 4. Voo, K.S. and Jacobsen B.M., Biotechniques 24, 240–243 (1998). 5. Hilz, H. et al. Eur. J. Biochem. 56, 103–108 (1975). 6. Ausubel, F.M. et al., eds., Current Protocols in Molecular Biology 1, 1.68 (1991). 7. Melzak, K. A. et al., J. Coll. Interf. Sci. 181, 635–644 (1996).
M U 1 2 3 4 Real-time PCR amplification from genomic DNA extracted from K3 EDTA-treated whole blood using the illustra blood genomicPrep Mini Spin Kit. The eluted product (3 µl ) and various amounts (0.1, 1, 10, and 100 ng) of control DNA were amplified using chromosome-specific primers in a real-time PCR assay on an ABI 7900. Replicates were n = 3.
PAGE: 155 - CURRENCY: USD
PAGE: 155 - CURRENCY: USD
The purification process involves minimal shearing resulting in the production of 5 to 10 µg of good quality, intact genomic DNA from a 200 µl sample. Input sample volumes can vary from 50 µl to 1 ml. The method uses a simple genomic DNA purification protocol that uses chaotropic agents to extract DNA from blood cells, denature protein components, and promote the selective binding of DNA to a column-based, novel silicamembrane. The elution volume can be modified as necessary to obtain the appropriate genomic DNA concentration for most downstream molecular biological applications. The kit contains spin columns prepacked with a novel silica membrane, lyophilized proteinase K powder, lysis solution, wash and elution buffers, microcentrifuge collection tubes, a full protocol booklet, and a detachable, quick reference protocol card.
ORDERING INFORMATION
Digestion of purified genomic DNA with restriction enzymes. Purified genomic DNA from the blood genomicPrep Mini Spin Kit was digested with AluI, EcoRI, HindIII, and BamHI restriction enzymes.
Kit compatibility across multiple anticoagulants. Isolation of genomic DNA was carried out from 200 µl of human (Hu) whole blood treated with K3-EDTA (EDTA), sodium citrate (Cit) and sodium heparin (Hep). The plot shows mean values for yield and purity with standard deviations.
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 155 - CURRENCY: USD
J For the rapid and reproducible isolation of high-quality genomic DNA from whole blood, buffy coat, bone marrow, and nucleated red blood cells. J High-quality genomic DNA with a greater proportion of higher molecular weight DNA and less shearing. J Demonstrated application to various types of whole blood including human, horse, rabbit, rat and mouse. J Increased ease of use with reduced pipetting volume changes, one centrifugation setting, and color-coded kit components. J Validated in several downstream applications including realtime PCR, endpoint PCR, multiplex PCR, and restriction enzyme digests.
155
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Nucleic Acid Sample Preparation Genomic DNA Preparation illustra blood genomicPrep Midi Flow Kit
3
4
5
25
28-9042-61
illustra blood genomicPrep Midi Flow Kit
100
28-9042-62
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Master Mix
page 174
illustra Hot Start Mix RTG
page 173
illustra blood genomicPrep Mini Spin Kit RT-PCR Master Mix (2X)
page 155 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs ExoSAP-IT
page 188 page 183
illustra GenomiPhi HY DNA Amplification Kit
page 161
illustra GenomiPhi V2 DNA Amplification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 160 page 181
illustra GFX 96 PCR Purification Kit illustra AutoSeq G-50
page 182 page 184
TECHNICAL SPECIFICATIONS blood Midi (1 to 8 ml; 5 ml optimum) Yield Purity Total time of prep* Hands-on time* Size
85 to 120 µg 1.7 to 1.9 2h 45 min y 50 kb
*including lysis, purification & desalting
1
2
3
4
5 6
7 8
9 10 11 12 13 14 15 16 17 18 19
6 97 kb 48 kb 23 kb
PAGE: 156 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
2
Quantity Code Number
illustra blood genomicPrep Midi Flow Kit
Lanes 1, 3, 5 represent the samples digested with EcoRI for 2 h. Lanes 2, 4, 6 represent genomic DNA used in the digestion experiments. Pulse-field gel electrophoresis showing high-quality genomic DNA isolated from human whole blood using the illustra blood genomicPrep Midi Flow Kit and a QIAGEN blood & cell culture midi kit. Lanes 1 and 2 are lambda and low-range markers, Lanes 3 to 14 represent genomic DNA purified with the illustra blood genomicPrep Midi Flow Kit, and Lanes 15 to 19 represent genomic DNA purified with a Qiagen blood & cell culture midi kit.
For more information, visit www.gelifesciences.com
PAGE: 156 - CURRENCY: USD
1
Product
PAGE: 156 - CURRENCY: USD
156
The illustra blood genomicPrep Midi Flow Kit can be used to purify genomic DNA from human whole blood, buffy coat, and animal blood such as horse, sheep, rat, mouse, guinea pig, and chicken whole blood. The kit provides yields of up to 250 µg of high-quality genomic DNA. The kit is robust, highly reproducible, and versatile allowing you to choose either gravity or spin methods with input sample volumes of 1 to 8 ml. The genomic DNA isolated with the illustra blood genomicPrep Midi Flow Kit is suitable for most biological applications including PCR, realtime PCR, restriction enzyme digest, and sequencing. The kit uses a simple and efficient three-step process involving a twostep lysis procedure at ambient temperature, anion-exchange column chromatography, and a highly reproducible desalting process. The kit contains fast-flow genomic 250 columns, lyophilized proteinase K powder, lysis solutions, load and elution buffers, NAP-25 desalting column, spin column adapters, a full protocol booklet, and a detachable, quick reference protocol card.
ORDERING INFORMATION
PAGE: 156 - CURRENCY: USD
5
J For the rapid extraction and purification of high-quality genomic DNA from whole blood, buffy coat, and animal blood. J Shorter total DNA extraction time of 2 h and less hands on time—the inclusion of NAP-25 desalting column removes the need for time consuming and complicated isopropanol precipitation. J Genomic DNA of high molecular weight validated in several downstream applications including real-time PCR, endpoint PCR, Multiplex PCR, and restriction digest. J Increased ease of use with room temperature lysis, 3 purification steps, and color-coded kit components. J Versatile kit allows you to take advantage of our high capacity, pre-equilibrated fast flow resin columns in spin or gravity mode.
GE Healthcare
ch05.fm Page 157 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Genomic DNA Preparation illustra bacteria genomicPrep Mini Spin Kit
The kit contains illustra bacteria genomicPrep Mini spin columns prepacked with novel silica membrane, lyophilized proteinase K powder, lysis solutions, wash and elution buffers, 2 ml microcentrifuge collection tubes, a full booklet with a detachable, quick reference protocol card.
Product
50
28-9042-58
illustra bacteria genomicPrep Mini Spin Kit
250
28-9042-59
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Master Mix illustra Hot Start Mix RTG
page 174 page 173
illustra tissue and cells genomicPrep Midi Flow Kit RT-PCR Master Mix (2X)
page 154 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs
page 188
ExoSAP-IT illustra GenomiPhi HY DNA Amplification Kit
page 183 page 161
illustra GenomiPhi V2 DNA Amplification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 160 page 181
illustra GFX 96 PCR Purification Kit illustra AutoSeq G-50
page 182 page 184
TECHNICAL SPECIFICATIONS bacteria Mini (1 u 109 to 4 u 109 cells) Yield Purity Total time of prep* (gram negative) Hands-on time* Total time of prep* (gram positive) Hands-on time* Size
4 to 12 µg 1.7 to 1.9 40 min 15 min 55 min 15 min y 20 kb
*includes testing on a wide range of strains
References 1. Human Genome Resources [Online] http://www.ncbi.nlm.nih.gov/genome/guide/human 2. Sambrook, J. and Russell, D. W. Molecular Cloning, A Laboratory Manual, chapter 6 (2001). 3. Vogelstein, B. and Gillespie, D. Proc. Natl. Acad. Sci. USA 76, 615 (1979).
Restriction enzyme digestion of gDNA purified using illustra bacteria genomicPrep Mini Spin Kit and QIAamp DNA Mini Kit. Purified samples containing 250 ng of gDNA were used for digestion reactions with 20 to 80 units of enzyme at either 37°C for 18 hours. The results were visualized on 0.8% agarose gel. U = undigested (no enzyme) gDNA; B = BamHI; E = EcoRI; H = HindIII; M = 1 kb molecular weight marker.
GE Healthcare
Quantity Code Number
illustra bacteria genomicPrep Mini Spin Kit
PAGE: 157 - CURRENCY: USD
PAGE: 157 - CURRENCY: USD
PAGE: 157 - CURRENCY: USD
The illustra Bacteria genomicPrep Mini Spin Kit can be used to isolate genomic DNA from a wide range of bacterial strains such as DH5a, TOP10, JM109, Bacillus subtilis etc. The kit produces highly pure and intact genomic DNA with OD260/280 values of 1.75-1.85. You can use different elution volumes to isolate genomic DNA at concentrations suitable for most downstream applications including restriction enzyme digestion, RT-PCR, and long PCR. The method uses a lysis solution in combination with proteinase K to gently release genomic DNA into solution from bacteria cells. The DNA is deproteinated in the extraction solution and the genomic DNA is then bound onto a silica column in the presence of chaotropic solution. Contaminants are removed during wash steps and the purifed DNA is eluted with prewarmed TE solution.
ORDERING INFORMATION
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 157 - CURRENCY: USD
J For the rapid extraction and purification of high-quality genomic DNA from gram-positive and gram-negative bacteria. J Total DNA extraction time under one hour with reduced lysis time. J Can be used to purify 4 to 8 µg of genomic DNA from 2 u 10 9 bacteria cells with input amounts ranging from 1 u 109 to 4 u 109 bacteria cells. J Spin columns-prepacked with a silica membrane-provide excellent reproducibility and consistency. J Validated in several downstream applications including RT PCR, long PCR, and restriction digest. J Color-coded caps for ease of use.
157
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Nucleic Acid Sample Preparation Genomic DNA Preparation Introduction to Nucleon DNA Extraction Kits Nucleon DNA extraction kits were developed around the proprietary Nucleon resins that bind proteins and polysaccharides forming a semi-solid barrier during partitioning, facilitating removal of DNA in the aqueous phase and ensuring excellent DNA recovery. SELECTION GUIDE BY APPLICATION – Nucleon Extraction Kits Recommended Appliations
Nucleon HT
t t t t t
t t t t t
t t t t t
t t t t
Whole blood or cultured cells
Whole blood or cultured cells
Whole blood
Paraffin embedded and hard tissue
Nucleon PhytoPure (50 x 0.1 g)
Nucleon PhytoPure (50 x 1.0 g)
t t
t t
t t Plant
t t Plant
O
O Si O Si (CH2)3N = CH(CH2)3CHO O O Si O Si (CH2)3N = CH(CH2)3CHO O O
+H2N -
PROTEIN
+H2N -
PROTEIN
DNA containing upper phase
Solid stratum of Nucleon resin with bound proteins
Protein containing organic phase PROTEIN
PROTEIN
Formation of semi-solid stratum after addition of Nucleon resin and chloroform.
Mechanism of Nucleon resin protein binding.
SELECTION GUIDE BY APPLICATION – Nucleon Extraction Kit Components Components Code Number Nucleon Resin Phytopure Resin Reagent 1 Reagent 2 Reagent 3 Reagent 4 Reagent A Reagent B 5 M Sodium Perchlorate Proteinase K
Nucleon BACC1
Nucleon BACC2
Nucleon BACC3
Nucleon HT
RPN8501 8 ml
RPN8502 16 ml
RPN8512 16 ml
RPN8509 8 ml
220 ml 18 ml 6 ml
420 ml 110 ml 26 ml
510 ml (4x) 110 ml 26 ml
Nucleon PhytoPure (50 x 0.1 g)
Nucleon PhytoPure (50 x 1.0 g)
RPN8510
RPN8511
6 ml 31 ml 11 ml
12 ml 245 ml 85 ml
18 ml 6 ml 10 mg
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 158 - CURRENCY: USD
O O Si O Si (CH2)3N = CH(CH2)3CH = N O O Si O Si (CH2)3N = CH(CH2)3CH = N O O
PAGE: 158 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
Nucleon BACC3
PAGE: 158 - CURRENCY: USD
158
Nucleon BACC2
PAGE: 158 - CURRENCY: USD
5
DNA Sequencing PCR Restriction Digests Blotting Hybridization AFLP/RFLP RAPD Starting Material
Nucleon BACC1
ch05.fm Page 159 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Genomic DNA Preparation Nucleon BACC Genomic DNA Extraction Kits ORDERING INFORMATION Product
Quantity Code Number
Nucleon BACC1
1 kit
RPN8501
Nucleon BACC2 Nucleon BACC3
1 kit 1 kit
RPN8502 RPN8512
PCR amplification of blood and cell culture DNA purified using Nucleon BACC. Lane 1: 100 bp ladder, lane 2: negative template control, lanes 3–5: human blood DNA, lanes 6–8: HeLa cell DNA. PCR primers: Mouse methyltransferase gene. Separation on a 1.5% agarose gel.
Nucleon PhytoPure Genomic DNA Extraction Kits
PAGE: 159 - CURRENCY: USD
ORDERING INFORMATION Product
Quantity Code Number
Nucleon PhytoPure
50 preps u 0.1 g
RPN8510
Nucleon PhytoPure
50 preps u 1.0 g
RPN8511
For pricing information, visit www.gelifesciences.com/orderonline
J PhytoPure resin binds plant polysaccharides ensuring high yields of high-quality plant genomic DNA in only one hour. J Two-kit sizes provide economical scale-up using the same phenol-free protocol.
PAGE: 159 - CURRENCY: USD PAGE: 159 - CURRENCY: USD
J Developed for rapid extraction of high quality, high molecular weight genomic DNA from blood and cell cultures. J Features a safe, phenol-free protocol requiring only 30 min to complete. J Available for three extraction scales: - Nucleon BACC1 for 50 preps of 1 ml of whole blood or cultured cells (1 to 3 x 106 cells/ml). - Nucleon BACC2 for 50 preps of 10 ml of whole blood or cultured cells (3 to 10 u 106 cells/10 ml) 100 preps of 1 ml of whole blood or cultured cells (1 to 3 x 106 cells/ml). Note: Additional Reagent A will need to be prepared to process all 50 x 10 ml preps. - Nucleon BACC3 for 50 preps of 10 ml of whole blood (kit contains all reagents necessary for 50 x 10 ml blood extractions).
PLANT SPECIES – PhytoPure successfully removed DNA
RAPD analysis of 4 species using operon primer OPA 10. DNA diluted to approximately 25 ng per lane and run on a 1.2% agarose gel in TBE. Lane 1: 1 kb ladder; lane 2: willow DNA extracted by Nucleon PhytoPure; lane 3: willow DNA extracted by SDS/Phenol; lane 4: maize DNA extracted by Nucleon PhytoPure; lane 5: maize DNA extracted by SDS/Phenol; lane 6: rye DNA extracted by Nucleon PhytoPure; lane 7: rye DNA extracted by SDS/Phenol; lane 8: rhododendron DNA extracted by Nucleon PhytoPure; lane 9: rhododendron DNA extracted by SDS/Phenol; lane 10: 1 kb ladder. Note the complete failure of the amplification reactions with willow DNA extracted by the SDS/phenol.
Arabidopsis thaliana Brassica oleracea Brassica napus Capsicum annuum Capsicum frutescens Cereals (barley, maize, rye, wheat) Cocos nucifera Helianthus annus Helianthus tuberosus Hevea braziliensis Irvingia gabonensis Lotus japonicus
Lycopersicon esculentum Musa spp Malus spp Nicotiana tabacum Phaseolus vulgaris Pinus sylvestris Pisum sativum Rhododendron spp Salix spp Solanum tuberosum Swietenia macrophylla Sphagnum, bog moss
Nucleon HT Genomic DNA Extraction Kit J Facilitates the simple, phenol-free extraction of genomic DNA from paraffin-embedded sections and hard tissue requiring proteinase K digestion. J Each kit is designed to recover high-quality DNA from 50 preparations of up to 25 mg of hard tissue.
GE Healthcare
ORDERING INFORMATION Product Nucleon HT
Quantity Code Number 1 kit
RPN8509
For pricing information, visit www.gelifesciences.com/orderonline
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 159 - CURRENCY: USD
For pricing information, visit www.gelifesciences.com/orderonline
159
ch05.fm Page 160 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Genomic DNA Preparation illustra GenomiPhi V2 DNA Amplification Kit J Quick mini-scale genomic DNA preparation: 4 to 7 µg in 1.5 h. J No DNA amplification in no-template controls. J One simple protocol for all different types of source material. J Representative amplification of the whole genome. J High-quality DNA for PCR, restriction enzyme digestion, hybridization, cloning, array CGH high-throughput genotyping, and DNA archival. 1 µl (1–10 ng) input DNA
9 µl sample buffer
ORDERING INFORMATION Product
Quantity Code Number
illustra GenomiPhi V2 DNA Amplification Kit illustra GenomiPhi V2 DNA Amplification Kit illustra GenomiPhi V2 DNA Amplification Kit
25 reactions
25-6600-30
100 reactions 25-6600-31 500 reactions 25-6600-32
For pricing information, visit www.gelifesciences.com/orderonline Custom options of the product are available. Please contact your local sales representative or Technical Support to request. Larger pack sizes available on request; please inquire. For licensing information, see back of catalog. Related Products
page 159 page 175
7
Yield (µg)
Incubate at 30 °C for 1.5 h. Inactivate enzyme at 65 °C for 10 min.
4–7 µg product
6
10 ng DNA
5
No DNA
4 3 2
Schematic of the illustra GenomiPhi V2 DNA Amplification Kit protocol.
illustra GenomiPhi V2 DNA Amplification Kit is part of the Phi29 DNA polymerase family of products from GE Healthcare, for mini-scale genomic DNA preparation.
0 0
0.5
1
1.5
2
Amplification time (h)
Microgram quantities of high-quality genomic DNA in less than two hours.
For more information, visit www.gelifesciences.com
PAGE: 160 - CURRENCY: USD
The protocol is very simple. A typical DNA yield of 4 to 7 µg DNA can be achieved in less than two hours with limited hands-on time. The average product length is y 10 kb. The starting material for GenomiPhi reactions can be purified DNA from any commercial kit or homebrew method, or nonpurified cell lysate. Source materials that can be used upstream from the GenomiPhi V2 kit, and validated downstream applications, are available at www.gelifesciences.com/genomiphi.
1
PAGE: 160 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
Nucleon BACC Genomic DNA Extraction Kits illustra PuReTaq Ready-To-Go PCR Beads
PAGE: 160 - CURRENCY: USD
160
9 µl reaction buffer 1 µl enzyme mix
page 165 page 155
PAGE: 160 - CURRENCY: USD
5
Heat or chemically denature.
Refer To
TempliPhi 100/500 Amplification Kit illustra blood genomicPrep Mini Spin Kit
GE Healthcare
ch05.fm Page 161 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Genomic DNA Preparation illustra GenomiPhi HY DNA Amplification Kit J Midi-scale genomic DNA preparation (40 to 50 µg) from nanograms of source material. J Less hands-on time compared to traditional isolation methods. J Simple, automation-friendly protocol with great reproducibility. J Representative amplification of the whole genome. J High-quality DNA for array CGH, high-throughput genotyping, DNA archival, PCR, restriction enzyme digestion, hybridization, and cloning.
ORDERING INFORMATION Product
Quantity Code Number
illustra GenomiPhi HY DNA Amplification Kit
25 reactions 25-6600-22
illustra GenomiPhi HY DNA Amplification Kit illustra GenomiPhi HY DNA Amplification Kit
100 reactions 1000 reactions
For pricing information, visit www.gelifesciences.com/orderonline Custom options of the product are available. Please contact your local sales representative or Technical Support to request. Larger pack sizes available on request; please inquire. For licensing information, see back of catalog. Related Products
page 165 page 155
Nucleon BACC Genomic DNA Extraction Kits illustra PuRe Taq PCR RTG
page 159 page 175
Heat or chemically denature.
5
60 50
Incubate at 30 °C for 4 h. Inactivate enzyme at 65 °C for 10 min.
Refer To
illustra TempliPhi 100/500 Amplification Kit v2 illustra blood genomicPrep Mini Spin Kit
Y ield (µg)
22.5 µl reaction buffer 2.5 µl enzyme mix
22.5 µl sample buffer
10 ng DNA No DNA
40 30 20 10 0
Schematic of the illustra GenomiPhi HY DNA Amplification Kit protocol.
The illustra GenomiPhi HY DNA Amplification Kit is part of the Phi29 DNA polymerase family of products from GE Healthcare. It contains all of the components necessary for genomic DNA preparation by isothermal strand displacement amplification.
0
1
Large quantities of high-quality DNA.
The product protocol is very simple, and provides typical yields of 40 to 50 µg DNA and average product lengths y 10 kb. The starting material for GenomiPhi reactions can be purified DNA or nonpurified cell lysates. Source materials that can be used upstream from the GenomiPhi HY kit, and validated downstream applications, are available at www.gelifesciences.com/genomiphi.
GE Healthcare
2 Amplification time (h)
PAGE: 161 - CURRENCY: USD
PAGE: 161 - CURRENCY: USD
PAGE: 161 - CURRENCY: USD
45 µg product
For more information, visit www.gelifesciences.com
3
4
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 161 - CURRENCY: USD
2.5 µl input DNA
25-6600-20 25-6600-25
161
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Nucleic Acid Sample Preparation Plasmid DNA Preparation illustra plasmidPrep Mini Spin Kit J For the rapid isolation of high and low copy number plasmid DNA. J Total preparation time of less than 10 min—representing a 50% reduction in protocol time. J Produces high-quality plasmid DNA with excellent reproducibility for use in cloning, restriction enzyme digestion, PCR amplification, and DNA sequencing. J Increased ease of use provided minimal changes in pipetting volume from one part of the protocol to the next, less centrifugation steps, color-coded kit components, and lack of organic solvents.
50
28-9042-69
illustra plasmidPrep Mini Spin
250
28-9042-70
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Master Mix illustra Hot Start Mix RTG
page 174 page 173
illustra plasmidPrep Midi Flow Kit RT-PCR Master Mix (2X)
page 163 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs
page 188
ExoSAP-IT illustra GFX PCR DNA and Gel Band Purification Kit
page 183 page 181
illustra GFX 96 PCR Purification Kit illustra AutoSeq G-50
page 182 page 184
TECHNICAL SPECIFICATIONS plasmidPrep Mini (1.5 mL; A600 = 2.5 to 3.0) Yield Purity A260/A280 Purity A260/A230 Total time of prep Hands-on time Size
6 to 9 µg y 1.8 y 1.7 z 9 min z 3 min 6.3 kb
plasmidPrep Mini (3.0 ml; A600 = 2.5 to 3.0)
Restriction enzyme digestion of a single DNA sample extracted with the illustra plasmidPrep Mini Spin Kit (400 ng, 5 units, 37°C for 1 h).
Yield Purity A260/A280 Purity A260/A230 Total time of prep Hands-on time Size
9 to 15 µg y 1.8 y 1.7 z 9 min z 3 min 6.3 kb
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References 1. Birnboim, H.C. and Doly, J., Nucl. Acids Res. 7, 1513 (1979). 2. Ish-Horowicz, D. and Burke, J.F., Nucl. Acids Res. 9, 2989 (1981). 3. Sambrook, J., et al., Molecular cloning: A laboratory Manual, Cold Spring Harbor laboratory, 2nd eds., (1989). 4. Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. USA 76, 615 (1979). 5. Marko, M.A., et al., Anal. Biochem. 121, 382 (1982). 6. Schoenfeld, et al ., Promega Notes 53, 12–19, (1995).
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 162 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
Quantity Code Number
illustra plasmidPrep Mini Spin
PAGE: 162 - CURRENCY: USD
162
Product
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5
The illustra plasmidPrep Mini Spin Kit uses a simple plasmid DNA purification protocol involving a modified alkaline lysis procedure and a novel silica-based membrane to achieve highly efficient plasmid DNA purification. The typical plasmid DNA yield from a fresh 1.5 ml culture of E. coli containing a high copy number plasmid is approximately 9 µg. Instead of organic solvents, the kit contains chaotropic salts to denature protein components and promote selective binding of DNA to the novel silica membrane. The kit contains spin columns prepacked with a novel silica-membrane, suspension, lysis, and neutralization buffers containing chaotropic salts, wash and elution buffers, microcentrifuge collection tubes, a full protocol booklet, and a detachable, quick reference protocol card.
ORDERING INFORMATION
ch05.fm Page 163 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Plasmid DNA Preparation illustra plasmidPrep Midi Flow Kit
The illustra plasmidPrep Midi Flow Kit can be used to process bacterial sample volumes of 25 to 50 ml for high copy number plasmid DNA and up to 500 ml for low copy number plasmid DNA. The kit consists of a high-performance, anion-exchange purification medium that combines excellent flow characteristics with an exceptional capacity and specificity for plasmid DNA. The kit contains fast-flow plasmid 250 columns, resuspension, lysis, neutralization, wash, elution, and TE buffers, and a full protocol booklet with a detachable, quick reference protocol card.
ORDERING INFORMATION Product
Quantity Code Number
illustra plasmidPrep Midi Flow
25
28-9042-67
illustra plasmidPrep Midi Flow
100
28-9042-68
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Master Mix
page 174
illustra Hot Start Mix RTG
page 173
illustra plasmidPrep Mini Spin Kit RT-PCR Master Mix (2X)
page 162 page 178
illustra Ready-To-Go RT-PCR Beads illustra PuReTaq Ready-To-Go PCR Beads
page 178 page 175
FideliTaq PCR Master Mix FideliTaq RT-PCR Master Mix
page 176 page 179
illustra Solution dNTPs ExoSAP-IT
page 188 page 183
NEW TempliPhi HT
page 161
illustra GFX PCR DNA and Gel Band Purification Kit illustra GFX 96 PCR Purification Kit
page 181 page 182
Cloning Kits illustra AutoSeq G-50
page 135 page 184
TECHNICAL SPECIFICATIONS Yield, purity, and quality values obtained with high and low copy number plasmid DNA purified with the plasmidPrep Midi Flow Kit.
PAGE: 163 - CURRENCY: USD
Plasmid copy number Plasmid size (kb) Culture volume (ml) Overall plasmid DNA yield (µg) Endotoxin (EU/µg pDNA) A260:A280 ratio A260:A230 ratio Supercoiled momomer (%) Transfection (% COS7 cells) Sequencing (Phred20 read length)
High 6.3 50 289 0.05 1.90 2.34 91.5 70 818
Low 36 150 191 0.13 1.93 2.29 ND ND ND
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plasmidPrep Midi (25 to 150 ml culture) Effective capacity Yield
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Agarose DNA electrophoresis showing uncut samples with predominantly supercoiled monomeric plasmid DNA and no endonuclease activity. The plasmid DNA was digested to completion with SacI and HindIII.
GE Healthcare
% supercoiled pDNA endotoxin Total time of prep Hands-on time Size
For more information, visit www.gelifesciences.com
y/= 250 µg 100 to y/= 250 µg high copy number plasmid; 20 to 250 µg low and very low copy number plasmid y 70% (strain dependent) v 10 EU per µg plasmid DNA z 215 min z 40 min 2 to y 50 kb
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 163 - CURRENCY: USD
J For the rapid extraction and purification of high-quality plasmid DNA from medium scale cultures for use in transfection, sequencing, and enzymatic amplification and modification. J High-column capacity allows for a proportional yield for input sample volume of 25 to 50 ml (for high copy number plasmid) and up to 500 ml for low copy number plasmid. J Provides plasmid DNA with excellent purity and low endotoxin levels. J Increased ease of use due to minimal pipetting volume changes, color-coded kit components, and pre-equilibrated columns.
163
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Nucleic Acid Sample Preparation Plasmid DNA Preparation Sephacryl S-1000 J For large-scale purification of plasmid DNA by gel filtration. J Yields highly pure plasmid DNA with no need for ultracentrifugation. J Heat stability allows sterilization by autoclaving. J Exceptional flow properties allow columns to be packed and equilibrated quickly and easily. J Can be used in aqueous buffer systems (pH 2 to 11), in concentrated urea or in guanidine hydrochloride, as well as in a number of organic solvents.
Quantity Code Number 750 ml
17-0476-01*
For pricing information, visit www.gelifesciences.com/orderonline * Availability varies by region. Contact local office for availabiity.
Sephacryl S-1000 Superfine gel filtration medium has a high exclusion limit. The inert and highly stable matrix, specially treated to provide high recoveries of biopolymers, is prepared by covalently cross-linking allyl dextran with N,N’-methylenebisacrylamide. The high mechanical strength of the resulting beads allows high flow rates and fast separations to be achieved.
Yeast Plasmid Isolation Kit J Eliminates the need to shuttle plasmids from yeast to E. coli. J Employs an enzymatic cell-wall removal method without the use of phenol/chloroform extraction or glass bead disruption. J Supplied with reagents that can be used to obtain yeast spheroplasts.
ORDERING INFORMATION Product Yeast Plasmid Isolation Kit
Quantity Code Number 50 reactions
US79220-50RXNS
For pricing information, visit www.gelifesciences.com/orderonline
This reagent kit is a mini-prep kit for yeast cells. It is designed for the isolation of 2-micron (µ) plasmids from yeast patches on plates or from yeast grown in small liquid culture.
PAGE: 164 - CURRENCY: USD
The isolated plasmid DNA can be used for PCR amplification, bacterial transformation or for direct sequencing. The ability to sequence directly from the isolated plasmid without the need for bacterial transformation or a pre-amplification step and restriction enzyme digestion makes it an efficient method for screening yeast transformants.
PAGE: 164 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
Sephacryl S-1000 SF
The Yeast Plasmid Isolation Kit contains the following components in sufficient quantities for performing 50 reactions from 1.5 ml cultures or 1 cm2 patches from plates: enzyme solution, spheroplast solution, lysis solution, precipitation solution, and protocol. Storage conditions: This kit and all enclosed reagents should be stored at -20°C in a non frost-free freezer. Safety precautions: Toxic and irritant.
For more information, visit www.gelifesciences.com
PAGE: 164 - CURRENCY: USD
164
Product
PAGE: 164 - CURRENCY: USD
5
ORDERING INFORMATION
GE Healthcare
ch05.fm Page 165 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Plasmid DNA Preparation illustra TempliPhi DNA Sequencing Template Amplification Kit / illustra TempliPhi 100/500 Amplification Kit
PAGE: 165 - CURRENCY: USD PAGE: 165 - CURRENCY: USD PAGE: 165 - CURRENCY: USD
Two different types of TempliPhi DNA Amplification Kits are available to meet different throughput requirements. illustra TempliPhi DNA Amplification Kit for 10 000 reactions contains premixed components and generates templates after overnight incubation. The 100- and 500-reaction kits contain separate, unmixed components for enhanced stability.
Product
10 000 reactions
25-6400-01
100 reactions
25-6400-10
500 reactions
25-6400-50
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
DYEnamic ET Dye Terminator Kit (MegaBACE)
page 225
DYEnamic ET Terminator Cycle Sequencing Kits Thermo Sequenase Dye Terminator Cycle Sequencing Kit
page 229 page 231
Thermo Sequenase II Dye Terminator Cycle Sequencing Kits illustra GenomiPhi V2 DNA Amplification Kit
page 230 page 160
2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 2
4
6
8
10
12
14
16
18
20
24
26
Kinetics of DNA amplification using the illustra TempliPhi 100 Amplification Kit. Amplification of 1 ng pUC19 DNA over 24 h. The amount of DNA was quantitated using PicoGreen dsDNA Quantitation Reagents at the given times. Data is representative of triplicate experiment.
Reaction Buffer
Enzyme Mix
Quantity
Storage
Quantity
Storage
Quantity
Storage
Quantity
Storage
100 Reaction Kit
1 x 0.5 ml
-20ºC (1 month) or 1 x 0.5 ml -70ºC (expiry on pkg)
-20ºC (1 month) or -70ºC (expiry on pkg)
1 x 20 µl
-20ºC (1 month) or -70ºC (expiry on pkg)
1 × 50 µl, 2 ng/µl
-20ºC (1 month) or -70ºC (expiry on pkg)
500 Reaction Kit
5 x 0.5 ml
-20ºC (1 month) or 5 x 0.5 ml -70ºC (expiry on pkg)
-20ºC (1 month) or -70ºC (expiry on pkg)
5 x 20 µl
-20ºC (1 month) or -70ºC (expiry on pkg)
1 x 50 µl, 2 ng/µl
-20ºC (1 month) or -70ºC (expiry on pkg)
Positive Control pUC19 DNA
TempliPhi Premix
Denature Buffer
Quantity
Storage
Quantity
Storage
Quantity
Storage
5 x 20 ml
-70˚C (6 months)
5 x 20 ml
-20˚C or -70˚C (12 months)
1 x 50 µl 2 ng/ul
-20˚C or -70˚C (12 months)
Positive Control pUC19 DNA
Components of each kit.
GE Healthcare
22
Time (hrs)
Sample Buffer
10 000 Reaction Kit
5
2.50
0
The illustra TempliPhi DNA Sequencing Template Amplification Kit has a distinct protocol and workflow compared to the 100/500 reaction size. Procedures for each kit are not interchangeable. Prior optimization is needed.
Quantity Code Number
illustra TempliPhi DNA Sequencing Template Preparation Kit illustra TempliPhi 100 Amplification Kit illustra TempliPhi 500 Amplification Kit
DNA yield (µg)
TempliPhi Kits use a novel process to efficiently prepare micrograms of circular DNA from picogram input material. The DNA templates are prepared by rolling circle amplification (RCA) using bacteriophage Phi29 DNA polymerase. TempliPhi uses an isothermal method for the exponential amplification of circular DNA. Phi29 DNA polymerase is active at 30°C, enabling amplification to be performed at this temperature without the need for thermal cycling. The TempliPhi protocol requires less than 20 min of hands-on time to amplify 96 samples from bacterial colonies.
ORDERING INFORMATION
For more information, visit www.gelifesciences.com
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 165 - CURRENCY: USD
J Prepare circular DNA templates for cycle sequencing, cloning, and transformation in 4 to 6 h. J Generate microgram quantities of template DNA from picogram amounts of starting material. J Use amplified DNA directly for cycle sequencing without purification. J Amplify DNA from bacterial or M13 liquid cultures, colonies, plaques, glycerol stocks, or purified circular (plasmid or M13) DNA. J Simple protocol reduces time, labor, and consumables needed for template preparation, and workflow enables easy automation.
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Nucleic Acid Sample Preparation Plasmid DNA Preparation illustra TempliPhi Sequence Resolver Kit J For the amplification of difficult templates for successful DNA sequencing. J Solve the most common sequencing problems such as repeats, sequencing stops, and compressions with a single kit. J Improve difficult template sequencing success with up to 820 bp Phred 20 read length. J Cultureless preparation from bacterial colonies, glycerol stock or puriried DNA, saves time and reagents. J Easy-to-use kit requires only 20 min hands-on time. J Amplified DNA can be directly used in sequencing reactions.
20 reactions 28-9035-29
50 reactions 28-9035-30
200 reactions
28-9035-31
For pricing information, visit www.gelifesciences.com/orderonline
Electropherogram showing comparative resolution of a GC-rich region with ABI PRISM dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit and the illustra TempliPhi Sequence Resolver Kit. Plasmid template was amplified with either the illustra TempliPhi 100 Amplification Kit or the illustra TempliPhi Sequence Resolver Kit. Amplified product (400 ng) was sequenced with either the ABI PRISM dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (illustra TempliPhi 100 product) or the DYEnamic ET Terminator Cycle Sequencing Kit (illustra TempliPhi Sequence Resolver product) according to their manufacturers' protocols. Sequencing reactions were analyzed on an ABI 3730xl DNA Analyzer. A GC-rich region toward the end of the sequence showed poor resolution when it was sequenced with dGTP chemistry—resulting in base miscalling. In addition, the dGTP chemistry produced sequencing compressions. All these limitations are absent from the sequence produced with the illustra TempliPhi Sequence Resolver Kit.
Comparative evaluation of of the illustra TempliPhi Sequence Resolver Kit and traditional resolution methods with various difficult sequence motifs1 illustra TempliPhi 100 Kit with additives to sequencing reactions DMSO (5%)
SequenceRx Enhancer A
Betaine (1 M)
GA dinuc repeat GC rich Poly G TG dinuc repeat CT dinuc repeat Poly C CA dinuc repeat/poly C TC TC at end of sequence Inverted repeat GC rich Inverted repeat
731 716 480 829 792 270 533 829 746 565 550 689
590 588 379 379 82 227 424 7 473 204 489 588
307 612 410 388 164 228 456 31 460 191 350 702
293 532 413 386 427 235 453 47 515 201 366 764
251 403 378 374 190 223 439 40 353 199 291 481
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Motif
illustra TempliPhi Sequence Resolver Kit None
1 Plasmid templates were amplified with either the illustra TempliPhi 100 DNA Amplification Kit or the illustra TempliPhi Sequence Resolver Kit. Amplified product (400 ng) was sequenced with the DYEnamic ET Terminator Cycle Sequencing Kit according to the manufacturer's protocol. The indicated reagents were added to the sequencing reactions for the illustra TempliPhi 100 amplified products. Sequencing reactions were analyzed on an ABI 3730xl DNA Analyzer, and Phred 20 read lengths (bp) were tabulated for all the samples. The highlighted samples are those that yielded the longest read length for each motif.
For more information, visit www.gelifesciences.com
PAGE: 166 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
Quantity Code Number
illustra TempliPhi Sequence Resolver Kit Z illustra TempliPhi Sequence Resolver Kit Z illustra TempliPhi Sequence Resolver Kit Z
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166
Product
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5
illustra TempliPhi Sequence Resolver Kit uses the highly processive, strand-displacing Phi29 DNA polymerase and modified nucleotides to amplify circular templates for subsequent sequencing. The kit can be used with both small (plasmids, M13) and large [fosmids and bacterial artificial chromosomes (BACs)] circular templates. The starting template for the reaction can be purified DNA, glycerol stock, liquid culture or colonies. The reaction takes less than 20 min to prepare and after an overnight incubation at 10°C, it is ready to be used directly in sequencing applications with the sequencing chemistry of choice. A ten microliter (10 µl) reaction typically yields 1 µg of DNA in a standard overnight reaction.
ORDERING INFORMATION
GE Healthcare
ch05.fm Page 167 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Plasmid DNA Preparation illustra TempliPhi Large Construct Kit ORDERING INFORMATION Product
Quantity Code Number
illustra TempliPhi Large Construct Kit
1000 reactions
25-6400-80
For pricing information, visit www.gelifesciences.com/orderonline Related Products
illustra GenomiPhi V2 DNA Amplification Kit DYEnamic ET Terminator Cycle Sequencing Kits
page 160 page 229
J Developed for high-throughput users and specifically designed for BAC or fosmid DNA preparation. J Start from microliter quantities of purified templates, as well as bacterial culture or glycerol stocks. J Generate 5 µg amounts of total DNA. J Simplified process, 20 min hands-on time. J Amplification is completed in 18 h with no need for thermal cycling.
Sample Buffer
Reaction Buffer
Enzyme Mix
Quantity
Storage
Quantity
Storage
Quantity
Storage
1 x 9 ml
-70ºC or -20ºC
1 x 9 ml
-70ºC or -20ºC
1 x 500 µl
-70ºC
Components of illustra TempliPhi Large Construct Kit.
PlasmidSelect Xtra Platform For main product entry, see page 497.
PAGE: 167 - CURRENCY: USD
PAGE: 167 - CURRENCY: USD
PAGE: 167 - CURRENCY: USD
TempliPhi Large Construct Kit 1000 reactions
page 165
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 167 - CURRENCY: USD
BAC with a 90-kb insert DNA sequencing result from 2 µl of glycerol stock using TempliPhi Large Construct Kit on ABI 3730 (BigDye v3.1, premix 8 µl per reaction).
Refer To
illustra TempliPhi DNA Sequencing Template Amplification Kit / illustra TempliPhi 100/500 Amplification Kit
167
ch05.fm Page 168 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation RNA Preparation illustra RNAspin Mini Isolation Kit Quantity Code Number
illustra RNAspin Mini Kit
20 preps
25-0500-70
illustra RNAspin Mini Kit illustra RNAspin Mini Kit
50 preps 250 preps
25-0500-71 25-0500-72
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Ready-To-Go RT-PCR Beads
page 178
RT-PCR Master Mix (2X) Amersham CyScribe First-Strand cDNA Labeling Kit
page 178 page 338
Amersham CyScribe Post-Labeling Kit
page 339
The illustra RNAspin Mini Isolation Kit can be used to isolate total RNA from cultured cells, tissue, bacteria or yeast. The isolated RNA is of a high enough quality and quantity (up to 100 µg) for multiple downstream experiments and sensitive enzymatic applications like RT-PCR, primer extension, and RNase protection assays. The RNAspin Mini Kit has been used for the isolation of high-quality total RNA from several types of source material including hard-to-lyse bacterial strains, and applied in downstream experiments such as quantitative reverse transcriptase PCR (QRT-PCR), Northern blot, and microarray experiments. The kit is supplied with a full protocol booklet with a detachable, quick reference protocol card, and all the necessary components including prefilters and DNase I. TECHNICAL SPECIFICATIONS Sample size Typical Yield Elution volume Effective binding capacity RNA integrity
Animal Tissue up to 30 mg tissue up to 70 µg
For more information, visit www.gelifesciences.com
sharp rRNA bands with no substantial degradative bands visible 28S:18S = z 2:1 RNA Integrity Number (RIN) values x 7 A260/A280 = 1.8–2.2 v 30 min/6 preps
GE Healthcare
PAGE: 168 - CURRENCY: USD
RNA purity Time/Prep
Cell Culture up to 5 u 106 cells up to 70 µg 40–120 µl 100 µg
PAGE: 168 - CURRENCY: USD
168
The illustra RNAspin Mini Kit produces high-quality RNA: rRNA bands are sharp, with the 28S band being about twice as intense as the 18S band, and with good RNA Integrity Number (RIN) values. (A) Total RNA from 106 HeLa cells was isolated with RNAspin Mini and separated by gel electrophoresis on a 1.2% formaldehyde agarose gel; (B) Total RNA from rat liver was isolated with RNAspin Mini and evaluated using the Agilent 2100 bioanalyzer.
Product
PAGE: 168 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
ORDERING INFORMATION
PAGE: 168 - CURRENCY: USD
J High-quality output RNA from diverse sample types; suitable for use in sensitive downstream applications. J Recovers high-quality total RNA due to the removal of genomic DNA through on-column DNase I treatment. J For maximum yield and purity, prefilters are included to reduce lysate viscosity. J Results can be obtained with even small amounts of precious sample (e.g., x 10 HeLa cells for RT-PCR). J Lysis buffer less susceptible to foaming to ensure valuable RNA sample is not wasted. J Simple and convenient format that is suitable for all levels of expertise.
ch05.fm Page 169 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation RNA Preparation illustra RNAspin Midi Isolation Kit
The illustra RNAspin Midi Isolation Kit can be used to isolate total RNA from cultured cells, tissue, bacteria, and yeast. The procedure is straightforward and it takes less than 30 min to complete. The isolated RNA is of sufficient quantity (up to 700 µg) and quality for downstream applications, including Northern blot analysis, quantitative reverse transcription polymerase chain reaction (QRT-PCR), primer extension or RNase protection assays. The kit is supplied with a full protocol booklet with a detachable, quick reference protocol and all the necessary components including prefilters and DNase I.
ORDERING INFORMATION Product
Quantity Code Number
illustra RNAspin Midi Kit
20 preps
Related Products
Refer To
Amersham AlkPhos Direct Labeling and Detection Systems Amersham Hybond-N+
page 331 page 326
Amersham Hybond-N
page 327
TECHNICAL SPECIFICATIONS Sample size Typical Yield Elution volume Effective binding capacity RNA integrity
Animal Tissue up to 200 mg tissue up to 700 µg
sharp rRNA bands with no substantial degradative bands visible 28S:18S = z 2:1 RNA Integrity Number (RIN) values x 7 A260/A280 = 1.8–2.2 80 min/4 preps
The illustra RNAspin Midi Kit produces high-quality RNA: rRNA bands are sharp, with the 28S band being about twice as intense as the 18S band, and with good RNA Integrity Number (RIN) values. (A) Total RNA from mouse liver was isolated with RNAspin Midi and separated by gel electrophoresis on a 1.2% formaldehyde agarose gel; (B) Total RNA from rat liver was isolated with RNAspin Midi and evaluated using the Agilent 2100 bioanalyzer.
PAGE: 169 - CURRENCY: USD
PAGE: 169 - CURRENCY: USD
Cell Culture up to 5 u 107 cells up to 700 µg 500 µl 700 µg
PAGE: 169 - CURRENCY: USD
RNA purity Time/Prep
GE Healthcare
25-0500-73
For pricing information, visit www.gelifesciences.com/orderonline
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 169 - CURRENCY: USD
J For applications requiring high yields. J High-quality output RNA from diverse sample types and it is suitable for use in sensitive downstream applications. J Scalable input and output. J High-quality total RNA due to the removal of genomic DNA through on-column DNase I treatment. J Prefilters included to remove lysate viscosity. This increases yield and purity with input tissues such as bacteria and yeast.
169
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Nucleic Acid Sample Preparation RNA Preparation illustra RNAspin 96 RNA Isolation Kit J For fast and efficient purification in a 96-well format. J 96 samples parallel-processed with high reproducibility in under 70 min. J Integrated wash plate eliminates risk of contamination. J Flexible processing by vacuum or centrifugation. J DNase I treatment removes genomic DNA leading to highquality total RNA product.
Quantity Code Number
illustra RNAspin 96 Kit
2 u 96 preps 25-0500-74
illustra RNAspin 96 Kit illustra RNAspin 96 Kit
4 u 96 preps 25-0500-75 24 u 96 preps 25-0500-76
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra RNAspin Mini Isolation Kit
page 168
illustra RNAspin Midi Isolation Kit illustra RNAspin 96 Filter Plate
page 169 this page
Animal Tissue Vacuum
RNA purity Time/Prep
10–30 mg up to 40 µg
Cell Culture Vacuum
30 mg tissue 2 u 106 cells up to 100 µg up to 20 µg 50–130 µl 100 µg
Centrifuge 1 u 107 up to 100
sharp rRNA bands with no substantial degradative bands visible 28S:18S = z 2:1 RNA Integrity Number (RIN) values x 7 A260/A280 = 1.8–2.2 70 min/96 preps
J Allows for tissue samples to be processed with the RNAspin 96 Kit. The RNAspin Filter plate is an accessory product that facilitates the use of tissues as input material for 96-well total RNA extraction with the RNAspin 96 Kit.
ORDERING INFORMATION Product illustra RNAspin 96 Filter Plate
Quantity Code Number 1 25-0500-88
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra RNAspin 96 RNA Isolation Kit
this page
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 170 - CURRENCY: USD
illustra RNAspin 96 Filter Plate
PAGE: 170 - CURRENCY: USD
The illustra RNAspin 96 Kit produces high-quality RNA: rRNA bands are sharp, with the 28S band being approximately double the intensity of the 18S band, as well as having high RNA Integrity Number (RIN) values. (A) Total RNA was purified from 10 mg of liver tissue using RNAspin 96. 100 µl of RNase-free water was dispensed onto the silica membrane for elution. 100 µl of RNA eluate was recovered by centrifugation, or 80 µl of RNA eluate was recovered using vacuum processing. Twenty microliters of each eluate was analyzed on a 1% formaldehyde agarose gel; (B) Total RNA from rat liver was isolated with RNAspin 96 centrifugation protocol, and 1 µl of a 100-µl eluate from six independent samples was evaluated using the Agilent 2100 bioanalyzer.
Sample size Typical Yield Elution volume Effective binding capacity RNA integrity
Centrifuge
PAGE: 170 - CURRENCY: USD
TECHNICAL SPECIFICATIONS
PAGE: 170 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
Product
The illustra RNAspin 96 Isolation Kit can be used to reproducibly isolate total RNA from cultured cells, tissue, bacteria or yeast. It allows for the simultaneous processing of 96 samples via either centrifugation or vacuum-based protocols that can easily fit onto existing automated systems. The isolated RNA is of a high enough quality and quantity (up to 100 µg) for multiple downstream experiments, including sensitive enzymatic applications like quantitative reverse transcription polymerase chain reaction (QRT-PCR), primer extension and RNase protection assays. Thus the RNAspin 96 kit is a flexible, highthroughput solution for total RNA isolation.
5
170
ORDERING INFORMATION
ch05.fm Page 171 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation RNA Preparation illustra Cesium Trifluoroacetate (CsTFA) J For separation and purification of nucleic acids, especially RNA, by isopycnic centrifugation. J Effective inhibitor of ribonucleases. J Can separate RNA and DNA. J Soluble in ethanol so nucleic acids can be recovered from the gradient by ethanol precipitation.
ORDERING INFORMATION Product illustra CsTFA (Solution)
Quantity Code Number 100 ml
17-0847-02
For pricing information, visit www.gelifesciences.com/orderonline
illustra CsTFA (solution*) is a strong chaotrope which not only inhibits ribonucleases, but also promotes the hydration and solubilization of nucleic acids and proteins. Due to its high solubility, solutions can be made with densities of up to 2.6 g/ml. This feature, combined with the fact that nucleic acids have relatively low buoyant densities in CsTFA (as compared with CsCl), means that RNA can be banded isopycnically in CsTFA gradients. Conditions can also be chosen in which RNA pellets at the bottom of the gradient while DNA is banded, thus allowing very effective purification of RNA from DNA.
5
PAGE: 171 - CURRENCY: USD
J For rapid purification of mRNA directly from a wide variety of eukaryotic cells or tissue samples, even from a single cell. J Fast and easy to use with no need for intermediate purification of total RNA. J Preserve integrity of mRNA by utilizing guanidine thiocyanate during crucial early stages of purification. J Utilize speed and selectivity of oligo(dT)-cellulose spin column chromatography to purify high-quality mRNA in yields suitable for direct use in cDNA synthesis and subsequent PCR, Northern blot hybridization and in vitro translation.
ORDERING INFORMATION Product
Quantity Code Number
illustra QuickPrep Micro mRNA Purification Kit
1 kit
27-9255-01
illustra QuickPrep mRNA Purification Kit (4 purifications)
1 kit
27-9254-01
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
Ready-To-Go You-Prime First-Strand Beads Ready-To-Go T-Primed First-Strand Kit
page 133 page 134
First-Strand cDNA Synthesis Kit illustra PuReTaq Ready-To-Go PCR Beads
page 133 page 175
The two illustra kits differ with respect to the number of purifications, the maximum amount of starting sample that can be used, the time required to purify mRNA and the prepackaging format of the kit components. Neither kit is applicable for mRNAs without poly(A+) tails (e.g., bacteria). The components in each kit are listed in the table below. * See licensing information at back of catalog.
PAGE: 171 - CURRENCY: USD
PAGE: 171 - CURRENCY: USD
illustra QuickPrep Micro mRNA Purification Kit* / illustra QuickPrep mRNA Purification Kit*
mRNA prepared from very small samples, then PCR-amplified following conversion to cDNA. Panel A: Rabbit b-globin cDNA. mRNA from a single rabbit reticulocyte was purified using QuickPrep mRNA Purification Kit, then reversetranscribed using First-Strand cDNA Synthesis Kit (27-9261-01) and pd(N)6 as primer. The entire reaction was amplified by PCR. Panel B: Radish RuBISCO cDNA. mRNA from a single primary leaf (lane 1) or from 100 mg of primary leaves (13 leaves) (lane 2) was purified using QuickPrep Micro mRNA Purification Kit, then reverse-transcribed using First-Strand cDNA Synthesis Kit and oligo(dT)12-18 as primer. The entire reaction was amplified by PCR using consensus RuBISCO primers.
GE Healthcare
For more information, visit www.gelifesciences.com
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 171 - CURRENCY: USD
*Density = 2.0 ± 0.05 g/ml
171
ch05.fm Page 172 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation RNA Preparation illustra mRNA Purification Kit Quantity Code Number
illustra mRNA Purification Kit 2 purifications (4 columns)
1 kit
27-9258-01
illustra mRNA Purification Kit 4 purifications (8 columns)
1 kit
27-9258-02
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra RNAspin Midi Kit illustra Ready-To-Go RT-PCR Beads
page 169 page 178
illustra PuReTaq Ready-To-Go PCR Beads
page 175
The illustra mRNA Purification Kit is designed to work smoothly in conjunction with QuickPrep Total RNA Extraction Kit or RNA Extraction Kit. Total RNA prepared with either kit can be applied directly to the oligo(dT)-cellulose spun columns with no intermediate precipitation required. Note: Not applicable for mRNAs without poly(A+) tails (e.g., bacteria). mRNA Purification Kit contains the following components predispensed for each purification: Oligo(dT)-cellulose columns; glycogen solution; protocol booklet; sample, high salt, low salt, and elution buffers. Synthesis of cDNA from mRNA prepared using RNA Extraction Kit (27-9270-01) followed by mRNA Purification Kit. Labeled cDNAs were synthesized using cDNA Synthesis Kit (27-9260-01) starting with 1 µg of mRNA from yeast (lane 1), HeLa cells (lane 2), calf liver (lane 3) or mung bean sprouts (lane 4).
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Product
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DNA, RNA, Protein Sample Prep & DNA Amplification
5
ORDERING INFORMATION
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J For rapid affinity purification of mRNA from eukaryotic total RNA using prepacked oligo(dT)-cellulose spun columns. J Purifies mRNA in 30 to 45 min from total RNA extracted from 25 mg to 1 g of cells or tissue. J Features predispensed reagents for each purification which minimizes the risk of nuclease contamination. J Produces mRNA ready for direct use (without precipitation) in procedures such as cDNA synthesis, PCR, Northern blot hybridization and in vitro translation. J Compatible with total RNA isolated by any currently available method.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 173 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components illustra Hot Start Mix RTG ORDERING INFORMATION Product illustra Hot Start Mix RTG
Quantity Code Number 0.5 ml tubes, 100 reactions
28-9006-46
illustra Hot Start Mix RTG
0.2 ml tubes, 96 reactions (12 u 8 28-9006-53 strip wells)
illustra Hot Start Mix RTG
0.2 ml tubes, 480 reactions (12 u 28-9006-54 8 strip wells u 5)
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
ExoSAP-IT
page 183
FideliTaq PCR Master Mix
page 176
illustra Hot Start Mix RTG uses a novel hot-start method called primer sequestration.
The Ready-To-Go format provides reproducible and reliable performance for demanding PCR applications in which high specificity and high sensitivity are essential to success. The mix does not contain Taq antibody and this eliminates the risk of mammalian-source contamination. Also, since the polymerase is not chemically-inactivated, there is no extensive heating step necessary which reduces the chance of damaging precious DNA samples from heat-induced depurination. Larger custom pack sizes are available on request, please inquire. * See licensing information at back of catalog.
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J Newly designed, premixed, predispensed reactions for Hot Start PCR featuring high-performance PuReTaq DNA polymerase. J Preformulated, predispensed, single-dose, ambienttemperature-stable beads ensure greater reproducibility between reactions, minimize pipetting steps and reduce the potential for pipetting errors and contamination. J Use of PuReTaq DNA polymerase and other high-purity reagents ensures that each bead is free of contaminating DNA. J Inhibition of primer-dimer formation, maximizes target amplification efficiency. J Eliminates nonspecific PCR product, primer sequestration blocks greater than 99% polymerase activity. J Improved performance in your experiments—greater than 99% accuracy in sequence amplification improves confidence in performing downstream applications.
illustra Hot Start Mix RTG combines high-quality recombinant Taq DNA Polymerase, a recombinant hot start activator protein, and nucleotides in a proprietary reaction buffer. The only additional reagents needed are water, template DNA, and primers. The RTG beads are provided in either 0.5 ml or 0.2 ml tubes that are compatible with most thermal cyclers. The 0.2 ml tubes come assembled in a 96-well (8 u 12) plate format that allows individual strips of eight tubes to be easily removed. This flexibility allows use of the entire 96-well plate, strips of eight, or individual 0.2 ml tubes.
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
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illustra Hot Start Mix RTG for PCR* requiring high specificity of amplification.
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Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components illustra Hot Start Master Mix J For PCR* requiring high specificity of amplification. J Inhibition of primer-dimer formation, maximizes target amplification efficiency. J Eliminates nonspecific PCR product, primer sequestration blocks greater than 99% polymerase activity. J Preserves sample integrity—milder PCR activation conditions maintain sample integrity compared to some other commercially available hot start methods. J Improved performance in your experiments—greater than 99% accuracy in sequence amplification improves confidence in performing downstream applications.
ORDERING INFORMATION Product illustra Hot Start Master Mix
Quantity Code Number 100 reactions 25-1500-01
For pricing information, visit www.gelifesciences.com/orderonline Shipped on dry ice. Store at -20°C. Mix well prior to use. Related Products
Refer To
ExoSAP-IT FideliTaq PCR Master Mix
page 183 page 176
illustra GFX 96 PCR Purification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 182 page 181 PAGE: 174 - CURRENCY: USD
illustra Hot Start Master Mix uses a novel hot start method called primer sequestration.
Each lot is functionally tested in a PCR such that inhibition of nonspecific amplification products is demonstrated. Hot Start Master Mix combines Taq DNA Polymerase with a recombinant hot start protein in a unique buffer formulation. Magnesium and nucleotide concentrations are 3 mM and 0.4 mM each, respectively. Larger custom pack sizes are available on request, please inquire. * For licensing information, see back of catalog.
Comparison of illustra Hot Start Master Mix to other commercially available hotstart systems in a polymerase blocking assay. Reactions in lanes 1 to 3 used PCR master mix, lanes 4 to 6 used Hot Start PCR Master Mix, lanes 7 to 9 used antibodyinhibited polymerase master mix and lanes 10 to 12 used a chemically inhibited polymerase master mix. Reactions in Panel A were incubated for 4 h at 25°C. Reactions in Panel B were heat-treated at 95°C for 2 min (lanes 4 to 9) or at 95°C for 15 min (lanes 10 to 12) to stop the respective inhibitory mechanisms of the master mixes. All reactions were resolved on a 15% polyacrylamide TBE-Urea denaturing gel and quantitated using ImageQuant software.
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174
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DNA, RNA, Protein Sample Prep & DNA Amplification
5
illustra Hot Start Master Mix combines high-quality recombinant Taq DNA Polymerase, a recombinant hot start activator protein, and nucleotides in a proprietary reaction buffer. This ready-to-use mix provides robust and reliable performance for demanding PCR applications in which high specificity and high sensitivity are essential to success. The mix does not contain Taq antibody and this eliminates the risk of contamination from a mammalian source. Also, since the polymerase is not chemically-inactivated, there is no extensive heating step necessary which reduces the chance of damaging precious DNA samples from heat-induced depurination.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 175 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components illustra PuReTaq Ready-To-Go PCR Beads J Newly designed, premixed, predispensed reactions for PCR featuring high-performance PuReTaq DNA polymerase. J Preformulated, predispensed, single-dose, ambient-temperature-stable beads ensure greater reproducibility between reactions, minimize pipetting steps and reduce the potential for pipetting errors and contamination. J Use of PuReTaq DNA polymerase and other high-purity reagents ensures that each bead is free of contaminating DNA. J Optimized for standard PCR, each bead yields a reaction containing z 2.5 units of PuReTaq DNA polymerase, 10 mM Tris-HCl, (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, stabilizers, and BSA. J Validated for real-time PCR with MGB Eclipse Probe Systems. native Taq cloned Taq
PuReTaq M
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low-DNA Taq 2
Product illustra PuReTaq Ready-To-Go PCR Beads (0.2 ml tubes/plate) illustra PuReTaq Ready-To-Go PCR Beads (0.2 ml tubes/plate)
Quantity Code Number 96 reactions
27-9557-01
5 u 96 reactions
27-9557-02
illustra PuReTaq Ready-To-Go PCR Beads (0.5 ml tubes)
100 reactions
27-9558-01
illustra PuReTaq Ready-To-Go PCR Beads (0.2 ml hinged tube with cap)
96 reactions
27-9559-01
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra GenomiPhi V2 DNA Amplification Kit
page 160
illustra GFX 96 PCR Purification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 182 page 181
100 Base-Pair Ladder
page 321
illustra Hot Start Mix RTG
page 173
illustra PuReTaq Ready-To-Go PCR Beads* are designed for performing standard PCR. The only additional reagents are water, template DNA and primers. The beads are provided in either 0.5 ml or 0.2 ml tubes that are compatible with most thermal cyclers. The 0.2 ml tubes come assembled in a 96-well (8 u 12) plate format that allows individual strips of eight tubes to be easily removed. This flexibility allows use of either the entire 96-well plate, strips of eight, or individual 0.2 ml tubes.
illustra PuReTaq Ready-To-Go PCR Beads use highly purified PuReTaq cloned Taq DNA polymerase. PCR amplifications were performed with two different commercial sources of “low DNA” Taq DNA polymerases; with native and cloned Taq DNA polymerases; and with PuReTaq DNA polymerase. Random primers were used without addition of template DNA. All reactions were amplified via standard PCR with 25 µl reactions: 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. Reactions were analyzed by gel electrophoresis with 10 µl loading volumes. Reproducibility of illustra PuReTaq Ready-To-Go PCR Beads versus original ReadyTo-Go PCR Beads in real-time testing. The threshold value represents the PCR cycle in which fluorescence increases above background value.
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* See licensing information at back of catalog.
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
M
low-DNA Taq 1
ORDERING INFORMATION
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Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components FideliTaq PCR Master Mix* J For PCR* requiring high-fidelity DNA polymerase activity. J Convenient, ready-to-use mix reduces experimental variability. J Optimized for long PCR products. FideliTaq PCR Master Mix* combines high-quality recombinant Taq DNA polymerase, a high-fidelity proofreading enzyme (3'-5' exonuclease), and Ultrapure nucleotides in a proprietary buffer formulation. FideliTaq PCR Master Mix Plus includes separate MgCl2 and PCR-qualified water.
Product
Quantity Code Number
FideliTaq PCR Master Mix (2u)
100 reactions (125 units)
E71182
FideliTaq PCR Master Mix Plus
100 reactions (125 units)
E71183
For pricing information, visit www.gelifesciences.com/orderonline Related Products FideliTaq DNA Polymerase illustra Hot Start Mix RTG
Refer To page 180 page 173
Sourced from E. coli expressing recombinant, thermostable DNA polymerases and free of detectable, nonspecific nucleases. Concentration is 0.05 units/µl. Store at -20°C. * For licensing information, see back of catalog.
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176
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DNA, RNA, Protein Sample Prep & DNA Amplification
5
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Functionally tested by producing a 20.7 kb PCR amplification product from lDNA.
ORDERING INFORMATION
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 177 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components illustra RAPD Analysis Beads
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Product
Quantity Code Number
illustra Ready-To-Go RAPD Analysis 100 reactions 27-9500-01 Beads illustra Ready-To-Go RAPD Analysis Kit 100 reactions and 6 primers 27-9502-01
For pricing information, visit www.gelifesciences.com/orderonline
5
RAPD analysis is a technique for rapidly detecting genomic polymorphisms. A single, short oligonucleotide primer of arbitrary sequence is used under low stringency conditions in PCR to generate a reproducible array of strain-specific products that are analyzed by gel electrophoresis. Under these conditions, genomic polymorphisms can be detected at multiple loci using only nanogram quantities of DNA. RAPD analysis has been used in numerous applications, including gene mapping, detection of strain diversity, population analysis, epidemiology and the analysis of phylogenetic and taxonomic relationships (2). The RAPD reactions are provided as dried beads that contain all the necessary PCR components—except primer—in concentrations optimized for RAPD analysis. Beads are provided in thin-walled 0.5 ml tubes compatible with most thermal cyclers. Each package of Ready-To-Go RAPD Analysis Beads contains sufficient reagents for 100 individual RAPD reactions: RAPD analysis beads, control E. coli BL21 DNA, control E. coli C1a DNA, RAPD analysis primer 2, and instruction booklet (DNA from two E. coli strains and RAPD analysis primer 2 are provided as controls to assay the ability of the RAPD beads to amplify DNA and identify polymorphisms.)
RAPD reactions for various organisms performed using 10 ng of template DNA, Ready-To-Go RAPD Analysis Beads and RAPD Analysis Primer 3 from the RAPD Analysis Primer Set.
The illustra RAPD Analysis Kit consists of the Ready-To-Go RAPD Analysis Beads and six primers which can be used with RAPD Analysis Beads. Each primer in the set is an arbitrary 10-mer which is specifically designed for use in RAPD analysis. The primers are supplied lyophilized and can be reconstituted with 500 µl of sterile distilled water to give a final concentration of 5 pmol/µl.
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ORDERING INFORMATION
References 1. Williams, J. G. et al. Nucl. Acids Res. 18, 6531 (1990). 2. Welsh, J. et al. PCR 2: A Practical Approach, McPherson, M. J., Hames. B. D. and Taylor, G. R., eds., Chapter 11, IRL Press (1995). * See licensing information at back of catalog.
Reproducibility of RAPD analyses using DNA extracted from E. coli BL21, Ready-To-Go RAPD Analysis Beads, and RAPD Analysis Primer 4. Reactions were performed on two days by two people (P1, P2) using two thermocyclers. M = 100 BasePair Ladder (27-4007-01).
GE Healthcare
For more information, visit www.gelifesciences.com
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 177 - CURRENCY: USD
J For performing DNA-profiling experiments using the randomly amplified polymorphic DNA [RAPD (1)] technique. J Preformulated, predispensed, single-dose reaction beads are provided as ambient-temperature-stable beads to ensure greater reproducibility between reactions, minimize pipetting steps and reduce the potential for pipetting errors and contamination. J RAPD* reactions are simple to perform—simply add genomic DNA solution and primer to a tube of RAPD Analysis Beads to a final reaction volume of 25 µl and cycle the reaction. J Each lot of RAPD Analysis Beads is function-tested to ensure its ability to generate a differential banding pattern between the two control E. coli strains using RAPD Analysis Primer 2 included with the RAPD beads. J RAPD reactions are pre-optimized for use with a wide variety of organisms.
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Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components illustra Ready-To-Go RT-PCR Beads J Preformulated, predispensed, single-dose, ambienttemperature-stable beads ensure greater reproducibility between reactions, minimize pipetting steps and reduce the potential for pipetting errors and contamination. J Optimized as a one-tube, one-step RT-PCR* reaction for both cDNA synthesis and PCR using only one buffer and primer set—no need to open the tube or change conditions between steps. J Each lot of RT-PCR Beads is function tested for its ability to generate highly specific PCR products to ensure lot-to-lot reproducibility.
illustra Ready-To-Go RT-PCR Beads (0.2 ml tubes) illustra Ready-To-Go RT-PCR Beads (0.2 ml hinged tube with cap)
Quantity Code Number 100 reactions 27-9266-01 96 reactions 96 reactions
27-9267-01 27-9259-01
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra RNAspin Mini Kit
page 168
illustra GFX 96 PCR Purification kit illustra QuickPrep Micro mRNA Purification Kit
page 182 page 171
Ready-To-Go You-Prime First-Strand Beads Ready-To-Go T-Primed First-Strand Kit
page 133 page 134
First-Strand cDNA Synthesis Kit MicroSpin S-400 HR Columns
page 133 page 182
illustra MicroSpin G-25 Columns
page 186
50 Base-Pair Ladder 100 Base-Pair Ladder
page 320 page 321
Homo-Oligomeric DNA
page 137
Ready-To-Go RT-PCR Beads have been optimized for RT-PCR. Each room-temperature-stable bead contains M-MuLV Reverse Transcriptase, RNase Inhibitor, Buffer, Nucleotides and Taq DNA polymerase. The only additional reagents required are water, template RNA, and primers. The Ready-To-Go Bead format significantly reduces the number of pipetting steps, thereby increasing reproducibility of the RT-PCR technique and minimizing risk of contamination and RNA degradation. Ready-To-Go RT-PCR Beads are provided in either thin walled 0.5 ml or 0.2 ml tubes compatible with most thermocyclers. The 0.2 ml tubes come assembled in a 96-well (8 u 12) plate format that allows individual strips of eight tubes to be easily removed. This flexibility allows use of either the entire 96-well plate, strips of eight or individual 0.2 ml tubes. Each package of Ready-To-Go RT-PCR Beads contains: RT-PCR beads, control reactions and pd(N)6 and oligo(dT) cDNA primers.
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DNA, RNA, Protein Sample Prep & DNA Amplification
illustra Ready-To-Go RT-PCR Beads (0.5 ml tubes)
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178
Product
* See licensing information at back of catalog.
RT-PCR Master Mix (2X) J For routine RT-PCR*. J Single-tube, one-step format minimizes contamination. J Convenient, ready-to-use mix reduces experimental variability. RT-PCR Master Mix (2X) combines all the reagents necessary for successful one-step RT-PCR. Suitable for amplifying targets up to 1.0 kb and free of detectable nonspecific nucleases. Functionally tested by amplifying a 459 bp b-actin target from 10 pg of human placental total RNA.
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5
illustra RT-PCR is commonly used to amplify cDNA by combining first-strand cDNA synthesis of an RNA template with PCR amplification. Typically the reaction is performed in two steps. A first-strand cDNA reaction is performed, the resulting cDNA is then transferred to another tube containing Taq DNA polymerase and PCR buffer where the reaction mixture is subjected to multiple cycles of denaturation, annealing and elongation, resulting in the exponential amplification of the target cDNA. Ready-To-Go RT-PCR Beads* simplify this process to a single-tube, single-reaction procedure.
ORDERING INFORMATION
ORDERING INFORMATION Product RT-PCR Master Mix (2X)
Quantity Code Number 100 reactions E78370
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
FideliTaq RT-PCR Master Mix (2X)
E71185
Refer To page 179
Supplied with RNase-free water and additional 25 mM MgCl2. Store at -20°C. * See licensing information at back of catalog.
Unique, proprietary formulation that includes M-MLV Reverse Transcriptase, Taq DNA Polymerase, recombinant Ribonuclease Inhibitor, Ultrapure nucleotides, and 3 mM magnesium in a novel RT-PCR buffer.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 179 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components FideliTaq RT-PCR Master Mix (2X) J For RT-PCR* requiring high-fidelity DNA polymerase activity. J Single-tube, one-step format minimizes contamination. J Convenient, ready-to-use mix reduces experimental variation. J Suitable for long PCR amplification (up to 6 kb). FideliTaq RT-PCR Master Mix (2X) combines all the reagents necessary for successful one-step RT-PCR. Suitable for amplifying targets up to 6 kb and free of detectable nonspecific nucleases. Functionally tested by amplifying a 459 bp b-actin target from 10 pg of human placental total RNA, a 1.5 kb b-actin target from 100 pg human placental total RNA and a 5.6 kb clathrin target from 10 ng pooled human total RNA.
ORDERING INFORMATION Product FideliTaq RT-PCR Master Mix (2X)
Quantity Code Number 100 reactions E71185
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
RT-PCR Master Mix (2X)
E78370
Refer To page 178
Unique, proprietary formulation that includes M-MLV Reverse Transcriptase, FideliTaq DNA Polymerase, recombinant Ribonuclease Inhibitor, nucleotides, and 3 mM magnesium in a novel RT-PCR buffer. Supplied with RNase-free water and additional 25 mM MgCl2. Store at -20°C. * See licensing information at back of catalog.
5
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Source: E. coli in which the gene for Thermus aquaticus is cloned. Unit definition: One unit incorporates 10 nmol of total nucleotides into acid-insoluble material in 30 min at 70°C in a total volume of 50 µl. Description: Taq DNA Polymerase (cloned) is the recombinant form of the native enzyme from Thermus aquaticus expressed in E. coli. Like native Taq, it polymerizes DNA from a primer annealed to a DNA template in the presence of deoxyribonucleotide triphosphates. Because Taq DNA Polymerase (cloned) is a recombinant protein, it is exceptionally pure and provides excellent batch-to-batch reproducibility. It has an optimum temperature of 75°C and can survive repeated incubations at y 95°C. Purity: Free from detectable nonspecific nuclease and Taq I restriction endonuclease activities.
ORDERING INFORMATION Product
GE Healthcare
Quantity Code Number
Taq DNA Polymerase (cloned)
250 units
27-0798-04
Taq DNA Polymerase (cloned) Taq DNA Polymerase (cloned)
4 u 250 units 10 u 250 units
27-0798-05 27-0798-06
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra Hot Start Mix RTG illustra GFX 96 PCR Purification Kit
page 173 page 182
illustra GFX PCR DNA and Gel Band Purification Kit
page 181
Concentration: 5000 units/ml. Storage conditions: 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, 50% glycerol, stabilizers. Store at -20°C. Functional testing: Tested in PCR. Supplied with 10u reaction buffer: 100 mM Tris-HCl, pH 9.0, 15 mM MgCl2 and 500 mM KCl. Also supplied with 25 mM MgCl2 solution. * See licensing information at back of catalog.
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J For PCR and other procedures requiring DNA polymerase activity at elevated temperatures.
For more information, visit www.gelifesciences.com
DNA, RNA, Protein Sample Prep & DNA Amplification
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Taq DNA Polymerase (cloned)*
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Nucleic Acid Sample Preparation PCR/RT-PCR Kits and Components FideliTaq DNA Polymerase* J For PCR* requiring high-fidelity DNA polymerase activity. J Compatible with either blunt-end or TA cloning procedures. J Suitable for long PCR amplification. Source: E. coli expressing recombinant, thermostable DNA polymerases. Description: FideliTaq DNA polymerase is a combination of high-quality recombinant Taq DNA polymerase and a highfidelity, proofreading enzyme (3'I5' exonuclease).
Purity: Free of detectable, nonspecific nucleases.
5
Storage conditions: 20 mM Tris-HCl, pH 8.5, 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 50% glycerol and stabilizers. Store at -20°C.
50 units
E71180V
FideliTaq DNA Polymerase
250 units
E71180W
FideliTaq DNA Polymerase FideliTaq DNA Polymerase
1000 units 5 u 250 units
E71180X E71180Y
FideliTaq DNA Polymerase
5000 units
E71180Z
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
FideliTaq PCR Master Mix (2u)
E71182
page 176
Refer To
FideliTaq PCR Master Mix Plus illustra Hot Start Mix RTG
E71183
page 176 page 173
illustra GFX 96 PCR Purification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 182 page 181
Assay conditions: The reaction mixture contains 25 mM TAPS, pH 9.3, 50 mM KCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol, 200 µM dNTPs, 250 µg/ml activated salmon sperm DNA and FideliTaq DNA Polymerase at 74°C for 10 min. Supplied with 10x reaction buffer: 100 mM Tris-HCl pH 8.6, 500 mM KCl, 15 mM MgCl2. Separate MgCl2 solution (25 mM) also provided. * For licensing information, see back of catalog.
Hot Tub DNA Polymerase* ORDERING INFORMATION
Source: Thermus ‘ubiquitous’. Description: A thermostable enzyme with an optimum temperature of 75°C and can withstand temperatures of up to 95°C. Unit definition: One unit incorporates 10 nmol of total nucleotides into acid-insoluble material in 30 min at 70°C in a total volume of 50 µl.
Product Hot Tub DNA Polymerase
Quantity Code Number 2500 units
T0333P
For pricing information, visit www.gelifesciences.com/orderonline
Storage conditions: 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, 50% glycerol and stabilizers. Store at -20°C. * See licensing information at back of catalog.
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Purity: Free from detectable nonspecific nuclease. The enzyme is greater than 95% pure by SDS-PAGE. Activity: 3 units/µl.
PCR Fragment Sizing and Mapping For main product entries, – ECL Direct Nucleic Acid Labeling and Detection System, see page 331. – Vertical Electrophoresis, see page 283. – Submarine Electrophoresis, see page 300. – Mini Vertical Units, see pages 285 – 286. – Standard Vertical Units, see page 287.
For more information, visit www.gelifesciences.com
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180
Functional testing: Production of a 20.7 kb PCR amplification product from lambda DNA.
Quantity Code Number
FideliTaq DNA Polymerase
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DNA, RNA, Protein Sample Prep & DNA Amplification
Concentration: 5 units/µl.
Product
PAGE: 180 - CURRENCY: USD
Unit definition: One unit incorporates 10 nmol of total nucleotides into acid-insoluble material in 30 min at 74°C in a total volume of 50 µl.
ORDERING INFORMATION
GE Healthcare
ch05.fm Page 181 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation DNA cleanup illustra GFX PCR DNA and Gel Band Purification Kit
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The illustra GFX PCR DNA and Gel Band Purification Kit is designed for the rapid purification and concentration of PCR products or DNA fragments ranging in size from 50 bp to 10 kb. This kit can be used to purify DNA from reaction volumes up to 100 µl or agarose gel slices up to 900 mg. The kit combines a versatile chaotropic buffer with a glass fibre matrix supported in a spin-column for the purification of DNA from both solution and agarose gel. Typical recoveries range from 60 to 80% for DNA fragments from agarose gel to as high as 95% for PCR products from solution. DNA purity is exceptional; 99.5% of contaminants are removed.
ORDERING INFORMATION Product illustra GFX PCR DNA and Gel Band Purification kit illustra GFX PCR DNA and Gel Band Purification kit
Quantity Code Number 100 purifications 28-9034-70 250 purifications 28-9034-71
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra MicroSpin G-25 Columns illustra GFX 96 PCR Purification kit
page 186 page 182
ExoSAP-IT illustra AutoSeq G-50
page 183 page 184
illustra NICK Columns illustra MicroSpin G-25 Columns
page 185 page 186
Hybond-N+ (15 u 10 cm)
page 326
Taq DNA Polymerase (cloned) illustra PuReTaq Ready-To-Go PCR Beads (0.5 ml tubes)
page 179 page 175
DNA Polymerization Mix dNTP Set (20 mM each A,C,G,T) 50 Base-Pair Ladder
page 188 page 320
100 Base-Pair Ladder KiloBase DNA Marker
page 321 page 321
The illustra GFX PCR DNA and Gel Band Purification Kit contains the following components in sufficient quantities: GFX columns, collection tubes and color-coded bottles of capture buffer, wash buffer, and two elution buffers (Tris-HCl and sterile water). GFX columns
Marker Marker 10 000 bp 9000 bp 8000 bp 7000 bp 6000 bp 5000 bp 4000 bp 3000 bp
Purified Marker
Purified Marker
1000 bp
A 910 bp PCR fragment (from the Tumor Protein P53 ORF) was purified using the illustra GFX PCR DNA and Gel Band Purification Kit. Both PCR and agarose gel band extractions are shown for the same fragment, for both 50 µl and 10 µl elution volumes. A260/280 readings were used to determine purity levels.
750 bp 500 bp 300 bp 150 bp
2000 bp 50 bp
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1000 bp
The versatile illustra GFX PCR DNA and Gel Band Purification Kit can be used to purify a broad range of DNA fragments. The DNA markers were loaded onto the column and purified according to the standard protocol.
A 910 bp PCR fragment (from the Tumor Protein P53 ORF) was purified using the illustra GFX PCR DNA and Gel Band Purification Kit. Both PCR and agarose gel band extractions are shown for the same fragment, for both 50 µl and 10 µl elution volumes. A260 readings were used to determine purity levels.
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
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J For the isolation and concentration of DNA fragments (50 bp to 10 kb) from PCR mixtures, DNA-containing agarose gel bands, enzyme-based DNA modifications, and restriction digestions. J Fast and easy to use method with less than 10 min hands-on time. J Flexible 10 to 50 µl elution volume for different DNA concentration needs. J Two choices of elution buffer provided to fit downstream applications. J Highly pure DNA is ready for direct use in sequencing, PCR, labeling, restriction enzyme digestion, and cloning. J Color-coded caps for ease of use.
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Nucleic Acid Sample Preparation DNA cleanup illustra GFX 96 PCR Purification Kit Quantity Code Number
illustra GFX 96 PCR Purification kit
10 u 96 well plates
28-9034-45
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra MicroSpin G-25 Columns
page 186
ExoSAP-IT illustra AutoSeq G-50
page 183 page 184
illustra NICK Columns illustra MicroSpin G-25 Columns
page 185 page 186
Hybond-N+ (15 u 10 cm) Taq DNA Polymerase (cloned)
page 326 page 179
illustra PuReTaq Ready-To-Go PCR Beads (0.5 ml tubes) DNA Polymerization Mix dNTP Set (20 mM each A,C,G,T)
page 175 page 188
50 Base-Pair Ladder
page 320
100 Base-Pair Ladder KiloBase DNA Marker
page 321 page 321
TECHNICAL SPECIFICATIONS Protocol
Elution volume (µl)
Yield (%)
Centrifugation
10 50 100
55 95 90
Vacuum
50 100
59 78
illustra MicroSpin S-400 HR Columns ORDERING INFORMATION Product
Quantity Code Number
MicroSpin S-400 HR Columns
50
27-5140-01
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
illustra MicroSpin S-200 HR Columns
27-5120-01
Refer To page 184
PAGE: 182 - CURRENCY: USD
J illustra MicroSpin S-400 HR Columns: For rapid purification of PCR products (x 200 bp) from unincorporated primers (w 32-mers) and nucleotides using spin-column chromatography. – Accommodates 25 to 50 µl for post-PCR cleanup prior to cloning or a second amplification reaction or 51 to 100 µl for all other applications. J Convenient: Prepacked with Sephacryl S-400 HR preequilibriated in TE buffer. J Ready to use: Requires less than 4 min from sample application to collection of purified product. J Tested in nickase, single- and double-stranded exonuclease and RNase assays.
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182
The illustra GFX 96 PCR Purification Kit contains the following components in sufficient quantities: instruction booklet, binding plates, wash plates, collection plates and color-coded bottles of capture buffer, wash buffer and and a choice of two elution buffers (Tris-HCl and sterile water).
Product
PAGE: 182 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
The illustra GFX 96 Purification Kit utilizes glass-fiber matrix technology in a 96-well format for highly efficient purification of PCR products. Fragments from PCR are captured by the matrix in the presence of a chaotropic salt, and contaminants are conveniently removed by washing the matrix with a buffered ethanol solution.
ORDERING INFORMATION
PAGE: 182 - CURRENCY: USD
J For the purification of up to 96 PCR products (0.1 to 10 kb) simultaneously in as little as 15 min. J High yields of pure DNA recovered in a small volume of water or a low-ionic strength buffer. J Typical recoveries are y 85% for PCR products 100 bp to 10 kb in length; salt removal typically y 99%. J Prepacked glass-fiber matrix 96-well plates are validated for use with the Macherey Nagel NucleoVac 96 vacuum manifold. J Avoids ethanol precipitations and hazardous organic extractions. J Purified DNA is ready for use in most applications, including fluorescent sequencing, microarrays, labeling, hybridization, ligation, and transformation.
Purification of a PCR product with a pre-spun illustra MicroSpin S-400 HR Column. A z 500 bp fragment was amplified using 25-mer primers. Following PCR, the reaction mixture was spiked with an excess of primer (500 pmol). The entire reaction (50 µl) was loaded onto a pre-spun illustra MicroSpin S-400 Column. Equivalent amounts of unpurified and purified reaction mixture were analyzed by electrophoresis. M = 100 Base-Pair Ladder (27-4007-01).
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 183 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation DNA cleanup illustra MicroSpin S-300 HR Columns Product
Quantity Code Number
illustra MicroSpin S-300 HR Columns
50
27-5130-01
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
illustra MicroSpin S-200 HR Columns
27-5120-01
Refer To page 184
Purification of PCR products with an illustra MicroSpin S-300 HR Column. A 129 bp fragment was amplified using 20-mer primers. Prior to PCR, the reaction mixture was spiked with an excess of primer (500 pmol). The 50 µl reaction was purified using an illustra MicroSpinS-300 Column. 10 µl of the purified reaction mixture was analyzed by electrophoresis. M = 100 Base-Pair Ladder (27-4007-01).
illustra MicroSpin G-25 Columns For main product entry, see page 186.
ExoSAP-IT ORDERING INFORMATION Product
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PAGE: 183 - CURRENCY: USD
ORDERING INFORMATION
Quantity Code Number
ExoSAP-IT
100 reactions
US78200
ExoSAP-IT
500 reactions
US78201
ExoSAP-IT
2000 reactions
US78202
For pricing information, visit www.gelifesciences.com/orderonline Store at -15 to -30°C.
PAGE: 183 - CURRENCY: USD
Related Products
Refer To
illustra GenomiPhi V2 DNA Amplification Kit illustra Solution dNTPs
page 160 page 188
Sequenase Version 2.0 DNA Sequencing Kits illustra Hot Start Mix RTG
page 235 page 173
illustra GFX 96 PCR Purification Kit
page 182
illustra GFX PCR DNA and Gel Band Purification Kit
page 181
J For rapid and efficient purification of PCR products. J Features two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, for the removal of unwanted deoxynucleotides and primers with no interference in downstream applications. J One-tube, one-step method with no sample loss. J Purified DNA is ready for immediate use in manual and automated sequencing. J Eliminates spin columns to decrease time and expense to purify PCR product. J Simple processing makes ExoSAP-IT ideal in robotics, replacing beads, filtrations, and plates. References Schematic diagram of the ExoSAP-IT method.
GE Healthcare
5
DNA, RNA, Protein Sample Prep & DNA Amplification
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J illustra MicroSpin S-300 HR Columns: For rapid purification of PCR products (x 100 bp) from unincorporated primers (w 20-mers) and nucleotides using spin-column chromatography. – Accommodates 25 to 50 µl for post-PCR cleanup prior to sequencing. – Useful for purification of alkaline-denatured plasmid DNA prior to sequencing. J Convenient: Prepacked with Sephacryl S-300 HR preequilibrated in TE buffer. J Ready to use: Requires less than 4 min from sample application to collection of purified product. J Tested in nickase, single- and double-stranded exonuclease and RNase assays.
1. Dugan, K. A. et al. J. Forensic Sci 47, 811–818 (2002). 2. Hanke, M. and Wink, M. BioTechniques 17, 858–860 (1994). 3. Mu, J. et al. Nature 418, 323–326 (2002). 4. Silva, Jr., W. A. et al. BioTechniques 30, 537–542 (2001). 5. Werle, E. et al. Nucleic Acids Res. 22, 4354–4355 (1994).
For more information, visit www.gelifesciences.com
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Nucleic Acid Sample Preparation DNA cleanup illustra AutoSeq G-50 J For rapid removal of unincorporated fluorescent dyeterminators from cycle sequencing reactions prior to automated sequencing. J Columns are prepacked with Sephadex G-50 DNA Grade F and pre-equilibrated in double-distilled water with 0.05% Kathon CG/ICP Biocide added as a preservative. J Columns that are ready to use enable sample application to collection of purified product in less than 4 min.
illustra AutoSeq G-50 illustra AutoSeq G-50
Quantity Code Number 50 columns
27-5340-01
250 columns 27-5340-02 1000 columns
27-5340-03
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra GFX 96 PCR Purification Kit
page 182
illustra AutoSeq G-50 consists of MicroSpin columns containing Sephadex G-50, pre-equilibrated in double-distilled water. This is important because even the small amount of salt in traditional buffers (such as TE buffer) can cause electrophoretic artifacts on salt-sensitive automated sequencers.
illustra MicroSpin G-50 Columns ORDERING INFORMATION Product illustra MicroSpin G-50 Columns illustra MicroSpin G-50 Columns
Quantity Code Number 50 250
27-5330-01 27-5330-02
For pricing information, visit www.gelifesciences.com/orderonline
Note: Not for use with ultrasalt-sensitive automated DNA Sequencers (e.g., ALFexpress II and ABI 377 model).
illustra MicroSpin S-200 HR Columns ORDERING INFORMATION Product illustra MicroSpin S-200 HR Columns
Quantity Code Number 50
27-5120-01
For pricing information, visit www.gelifesciences.com/orderonline
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 184 - CURRENCY: USD
J For rapid purification of labeled single-stranded or doublestranded DNA fragments x 50 bases in length in a volume of 51 to 100 µl using spin-column chromatography.
PAGE: 184 - CURRENCY: USD
J For rapid purification of labeled DNA (x 20 bases) from unincorporated labeled nucleotides in a volume of 25 to 50 µl using spin-column chromatography. J Columns are prepacked with Sephadex G-50 Grade preequilibrated in TE buffer with 0.05% Kathon CG/ICP Biocide. J Product recovery will be at least 10% lower than that from ProbeQuant G-50 Micro Columns. J Can also be used for buffer exchange/desalting of enzymatic reactions (10 to 100 µl) following heat denaturation and phenol extraction.
PAGE: 184 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
illustra AutoSeq G-50
illustra AutoSeq G-50 columns can be used to clean up reactions prepared with the BigDye Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems .
5
184
Product
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illustra AutoSeq G-50 columns are specifically designed to remove fluorescent dye-terminators from sequencing reactions prior to analysis on automated sequencers. Effective purification is essential for high-quality sequencing results since residual dye-terminators can obscure data.
ORDERING INFORMATION
ch05.fm Page 185 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation DNA cleanup illustra CyScribe GFX Purification Kit ORDERING INFORMATION Product
Quantity Code Number
illustra CyScribe GFX Purification Kit
25 purifications
27-9606-01
illustra CyScribe GFX Purification Kit
50 purifications
27-9606-02
For pricing information, visit www.gelifesciences.com/orderonline
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J Allows rapid purification of CyDye labeled cDNA probes. J Provides efficient removal of unincorporated CyDye label and primers from labeling reactions with superior yields of labeled cDNA probe. J Excellent for the purification of cDNA labeled by either direct incorporation or by post-labeling using CyScribe labeling kits. J Optimized purification reagents are conveniently available in both 25 and 50 column format or can be purchased with the CyScribe labeling kits.
Code Number
CyScribe First-Strand cDNA Labeling Kit
RPN6200
page 338
Refer To
CyScribe First-Strand cDNA Labeling System-dUTP
RPN6201
page 338
CyScribe First-Strand cDNA Labeling System-dCTP CyScribe Post-Labeling Kit
RPN6202 RPN5660
page 338 page 339
The illustra CyScribe GFX Purification Kit has been specifically developed for the purification of CyDye labeled cDNA probes. It can be used in conjunction with CyScribe microarray labeling kits for either direct incorporation of Cy3- and Cy5-labeled dNTPs or for post-labeling of cDNA with Cy3- and Cy5-monoreactive dye. The illustra CyScribe GFX Purification Kit uses a glass fiber matrix packed into a spin column format for highly efficient purification of CyDye labeled cDNA. Fluorescently labeled cDNA probes are captured by the matrix, and unincorporated CyDye and primer are removed by washing. Bound probes are eluted with a quick spin in elution buffer. High yields of purified fluorescent cDNA are ready for use in a variety of molecular applications, including hybridization and microarrays.
illustra NICK Columns J For rapid purification of labeled DNA (x 20 bases in length) from unincorporated radiolabeled nucleotides using gravity-flow chromatography. J Prepacked with Sephadex G-50 DNA Grade (see page 187) in distilled water with 0.15% Kathon CG/ICP Biocide. J Can accommodate sample volumes up to 100 µl.
ORDERING INFORMATION Product illustra NICK Columns illustra NICK Columns
Quantity Code Number 20 50
17-0855-01 17-0855-02
For pricing information, visit www.gelifesciences.com/orderonline
illustra ProbeQuant G-50 Micro Columns J For rapid purification of labeled DNA (x 20 bases) from unincorporated labeled nucleotides in a volume of 25 to 50 µl using spin-column chromatography. J Convenient: Prepacked with Sephadex G-50 DNA Grade (see page 187) and pre-equilibrated in STE containing 0.15% Kathon CG/ICP Biocide. J Ready to use: Requires less than 4 min from sample application to collection of purified product. J Designed for use in a microcentrifuge. J Tested in nickase, single and double-stranded exonuclease and RNase assays.
GE Healthcare
ORDERING INFORMATION Product illustra ProbeQuant G-50 Micro Columns
Quantity Code Number 50
28-9034-08
For pricing information, visit www.gelifesciences.com/orderonline Related Products Ready-To-Go DNA Labeling Beads (-dCTP)
For more information, visit www.gelifesciences.com
Refer To page 333
5
DNA, RNA, Protein Sample Prep & DNA Amplification
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Both Cy3 and Cy5 probes were prepared from human skeletal muscle total RNA using the CyScribe First-Strand cDNA Labeling Kit (RPN6200). The probes were purified using illustra CyScribe GFX cDNA spin columns and hybridized on a microarray slide spotted with human cDNA clones.
Related Products
185
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Nucleic Acid Sample Preparation DNA cleanup illustra MicroSpin G-25 Columns Product
50
27-5325-01
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
illustra GFX 96 PCR Purification Kit illustra GFX PCR DNA and Gel Band Purification Kit
page 182 page 181
TECHNICAL SPECIFICATIONS Volume* % Average yield† Standard deviation Average purity‡ Standard deviation *
100 µl
150 µl
83% 5.1 98% 2.9
89% 3.6 97% 4.8
A 17-mer was synthesized on a 0.2 µm scale and deprotected at 60°C for 30 min in concentrated ammonium hydroxide. 100 µl or 150 µl of deprotected oligo was applied to pre-spun MicroSpin G-25 Columns and centrifuged at 735 u g for 2 min in a microcentrifuge. As a standard, 0.25 ml of deprotected oligo was diluted to 1 ml and purified on a NAP-10 column according to instructions provided with the columns. Yield is based on A260 reading following purification. Yield from NAP-10 Column is a standard to which we have assigned 100% yield.
‡
Purity is based on amount of ammonia present following purification as determined using Nessler reagent. Purity from NAP-10 Column is a standard to which we have assigned 100% purity.
J For rapid and efficient purification of DNA and oligonucleotides (x 10-mers) in less than 15 min by gravity flow. J Prepacked with Sephadex G-25 DNA Grade in distilled water containing 0.15% Kathon CG/ICP Biocide. J Available in three sizes depending on sample volume: 0.5 ml (NAP-5), 1 ml (NAP-10) or 2.5 ml (NAP-25). J Useful for small-scale purification, desalting and buffer exchange.
ORDERING INFORMATION Product
Quantity Code Number
illustra NAP-5 Columns illustra NAP-5 Columns
20 50
17-0853-01 17-0853-02
illustra NAP-10 Columns illustra NAP-10 Columns
20 50
17-0854-01 17-0854-02
illustra NAP-25 Columns illustra NAP-25 Columns
20 50
17-0852-01 17-0852-02
For pricing information, visit www.gelifesciences.com/orderonline TECHNICAL SPECIFICATIONS Choosing a NAP column and buffer volumes Column Type
Sample Volume (ml)
Equilibration Buffer (ml)
Elution Buffer (ml)
NAP-5 Max. vol.
0.1 0.25 0.5
0.4 0.25 0
0.5 0.7 1.0
NAP-10 Max. vol.
0.75 1.0
0.25 0
1.2 1.5
NAP-25
1.5 2.0 2.5
1.0 0.5 0
2.5 3.0 3.5
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 186 - CURRENCY: USD
illustra NAP Columns
PAGE: 186 - CURRENCY: USD
†
Use of illustra MicroSpin G-25 Columns for desalting a DNA solution prior to restriction enzyme digestion. EDTA was added to pUC19 to a final concentration of 0 to 400 mM (as designated above each lane). Half of each 100 µl sample was applied to a MicroSpin. G-25 Column. Pre- and post-column samples were digested with HindIII. When HindIII was added to unpurified DNA containing 2.5 mM EDTA, digestion was incomplete; however, following column purification, even DNA with an original EDTA concentration of 400 mM was completely digested with HindIII. M = l DNA-HindIII Digest. (27-4048-01); con = control of uncut pUC19; pre = before column purification; post = after column purification.
Max. vol.
186
Quantity Code Number
illustra MicroSpin G-25 Columns
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DNA, RNA, Protein Sample Prep & DNA Amplification
ORDERING INFORMATION
PAGE: 186 - CURRENCY: USD
5
J For rapid buffer exchange/desalting of PCR products and other DNAs in a volume of 10 to 100 µl using spin-column chromatography. J Excellent for rapid purification of newly synthesized oligonucleotides x 10-mers in 100 to 150 µl of deprotection solution using spin-column chromatography. J Convenient: Prepacked with Sephadex G-25 DNA Grade and pre-equilibrated in distilled water containing 0.05% Kathon CG/ICP Biocide. J Ready to use: Requires less than 4 min from sample application to collection of purified product. J Tested in nickase, single and double-stranded exonuclease and RNase assays. J Can also be used for desalting/buffer exchange of DNA and removal of unincorporated radionucleotides from endlabeled oligonucleotides (at least 10 bases in length) in a volume of 10 to 100 µl.
ch05.fm Page 187 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation DNA cleanup illustra Sephadex G-25 DNA Grade J For purification of DNA (x 10 bases in length) from small molecules by gel filtration. J Tested to ensure reproducibly high recovery of DNA. J Used in MicroSpin G-25 Columns and NAP Columns. J When hydrated, 1 g of dry gel swells to 4 to 6 ml volume.
ORDERING INFORMATION Product illustra Sephadex G-25 DNA Grade SF
Quantity Code Number 100 g
17-0572-02
For pricing information, visit www.gelifesciences.com/orderonline
illustra Sephadex G-50 and G-100 DNA Grade ORDERING INFORMATION Product
25 g 100 g
17-0573-01 17-0573-02
illustra Sephadex G-100 DNA Grade SF
100 g
17-0574-02
For pricing information, visit www.gelifesciences.com/orderonline TECHNICAL SPECIFICATIONS
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DNA exclusion limit (base pairs) Oligonucleotide exclusion limit [poly (A)] ml of hydrated gel per gram of dry resin
GE Healthcare
Quantity Code Number
illustra Sephadex G-50 DNA Grade F illustra Sephadex G-50 DNA Grade F
For more information, visit www.gelifesciences.com
G-25
G-50
G-100
10 10 4–6
20 20 9–11
25 27 15–20
5
DNA, RNA, Protein Sample Prep & DNA Amplification
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J For purification of DNA from small molecules by gel filtration. J Tested to ensure reproducibly high recovery of DNA. J Sephadex G-50 DNA Grade is used in illustra AutoSeq G-50 columns, ProbeQuant G-50 Micro Columns, MicroSpin G-50 Columns, and NICK Columns. J Suitable for researchers who prefer to prepare their own columns for purifying nucleic acids.
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Nucleic Acid Sample Preparation Nucleotides illustra Solution dNTPs J Free from DNase, RNase, and nicking enzyme activity. J Greater than 99% triphosphate purity (by HPLC) for optimum performance and consistency. J Buffer free and ready-to-use solutions in multiple formats. J Functionally tested for long PCR* and sequencing. J Custom formulation, testing, packaging, and labeling are available.
A Certificate of Analysis for each lot is available on request.
High-purity nucleotides are functionally tested for long PCR. Lambda DNA (100 ng) was amplified with two primers 20.7 kb apart. PCR was performed under standard conditions using mixes (containing three dNTPs) spiked with the fourth dNTP, indicated above each set of three lanes. The volume of PCR product added is shown above each lane. The products were separated on a 0.8% agarose gel.
2'-Deoxyadenosine 5'-Triphosphate Solution, 100 mM
dCTP 2'-Deoxycytidine 5'-Triphosphate Solution, 100 mM
25 µmol 100 µmol
28-4065-01 28-4065-02
500 µmol
28-4065-03
25 µmol
28-4065-11
100 µmol 500 µmol
28-4065-12 28-4065-13
25 µmol 100 µmol
28-4065-21 28-4065-22
500 µmol
28-4065-23
dGTP 2'-Deoxyguanosine 5'-Triphosphate Solution, 100 mM
dTTP 2'-Deoxythymidine 5'-Triphosphate Solution, 100 mM
dUTP 2'-Deoxyuridine 5'-Triphosphate Solution, 100 mM
25 µmol
28-4065-31
100 µmol 500 µmol
28-4065-32 28-4065-33
25 µmol
28-4065-41
100 µmol
28-4065-42
dNTP Sets dNTP Set (100 mM each A,C,G,T)
4 u 25 µmol
28-4065-51
dNTP Set (100 mM each A,C,G,T) dNTP Set (100 mM each A,C,G,T)
4 u 100 µmol 4 u 500 µmol
28-4065-52 28-4065-53
DNA Polymerization Mix dNTP Set (20 mM each A,C,G,T)
10 µmol
28-4065-57
DNA Polymerization Mix dNTP Set (20 mM each A,C,G,T) PCR Nucleotide Mix dNTP Set (25 mM each A,C,G,T) PCR Nucleotide Mix dNTP Set (2 mM each A,C,G,T)
40 µmol (4 u 10 µmol)
28-4065-58
500 µl
28-4065-60
1 ml
28-4065-62
PCR Nucleotide Mix dNTP Set (10 mM each A,C,G,T)
500 µl
28-4065-64
For pricing information, visit www.gelifesciences.com/orderonline Custom formulation, testing, packaging, and labeling are available. Related Products Taq DNA Polymerase (cloned)
Refer To page 179
J For reducing “band compressions” in sequencing gels. J Supplied as convenient solutions at pH 7.0 and 7.5. c 7dGTP* are supplied in purified water as 5 and 10 mM solutions. Band compressions are ambiguous band patterns on sequencing gels caused by stable intra-strand secondary structures which are not fully denatured during electrophoresis. Their occurrence can be reduced by substituting nucleotide analogues in the sequencing mixes. To resolve G-band compressions, use 7-deaza dGTP (c7dGTP).
ORDERING INFORMATION Product 7-Deaza-2'-Deoxyguanosine 5'-Triphosphate, 5 mM Solution (c7dGTP)
Quantity Code Number 2 µmol 27-2090-02
For pricing information, visit www.gelifesciences.com/orderonline
References 1. Mizusawa, S. et al., Nucl. Acids Res. 14, 1319 (1986). 2. Barr, P. J. et al., BioTechniques 4, 428 (1986).
* See licensing information at back of catalog.
For more information, visit www.gelifesciences.com
GE Healthcare
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Deaza dGTP
PAGE: 188 - CURRENCY: USD
188
* See licensing information at back of catalog.
Quantity Code Number
dATP
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DNA, RNA, Protein Sample Prep & DNA Amplification
5
For information concerning molecular weights, structures, and extinction coefficients of representative nucleotides, see page 665.
Product
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By incorporating recent advances in manufacturing technology, GE Healthcare now offers illustra Solution dNTPs: the highest purity deoxynucleotides available for amplification, dideoxy sequencing, labeling, mutagenesis, cDNA synthesis, and expression profiling.
ORDERING INFORMATION
ch05.fm Page 189 Tuesday, June 17, 2008 11:47 PM
Nucleic Acid Sample Preparation Nucleotides High Purity Solution ddNTPs J Easy-to-use, time-saving solutions for use in any application requiring ddNTPs of high purity, such as dideoxy sequencing and genotyping reactions. J Each lot of ddNTPs is functionally tested to meet or exceed criteria for high-quality sequencing with Thermo Sequenase DNA polymerase. J High purity: x 98% triphosphate. J Available either as individual products or in a convenient set containing ddATP, ddCTP, ddGTP, and ddTTP in separate tubes. J Supplied in purified water as 5 mM and 100 mM solutions.
ORDERING INFORMATION Product ddNTP Set, 5 mM Solutions (ddATP, ddCTP, ddGTP, ddTTP) 2',3'-Dideoxyadenosine 5'-Triphosphate, 100 mM Solution (ddATP)
Quantity Code Number 4 u 0.5 µmol
27-2045-01
4 µmol (40 µl)
27-2051-01
2',3'-Dideoxycytidine 5'-Triphosphate, 100 mM Solution (ddCTP)
4 µmol (40 µl)
27-2061-01
2',3'-Dideoxyguanosine 5'-Triphosphate, 100 mM Solution (ddGTP) 2',3'-Dideoxythymidine 5'-Triphosphate, 100 mM Solution (ddTTP)
4 µmol (40 µl)
27-2071-01
4 µmol (40 µl)
27-2081-01
For pricing information, visit www.gelifesciences.com/orderonline
For information concerning molecular weights, structures and extinction coefficients of representative nucleotides, see page 665.
5
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J Easy-to-use, time-saving solutions for in vitro transcription or for reactions requiring rNTPs of high purity. J Each lot is tested for ribonuclease contamination to ensure success in your application. J High purity: x 98% triphosphate. J Supplied in purified water as 100 mM solutions (pH 7.5). J Available either as individual products or in a convenient set containing ATP, CTP, GTP, and UTP in separate tubes. J rNTPs are also available as solids for your convenience.
ORDERING INFORMATION Product
Quantity Code Number
rNTP Set rNTP Set, 100 mM Solutions (ATP, CTP, GTP, UTP) rNTPs, 100 mM Solutions Adenosine 5'-Triphosphate, 100 mM Solution (ATP)
4 u 25 µmol 27-2025-01
25 µmol 27-2056-01
Cytidine 5'-Triphosphate, 100 mM Solution (CTP)
25 µmol 27-2066-01
Guanosine 5'-Triphosphate, 100 mM Solution (GTP)
25 µmol 27-2076-01
Uridine 5'-Triphosphate, 100 mM Solution (UTP)
25 µmol 27-2086-01
rNTP Solids Adenosine 5'-Triphosphate, Disodium, Crystalline (ATP)
5 g 27-1006-01
Adenosine 5'-Triphosphate, Disodium, Crystalline (ATP) Cytidine 5'-Triphosphate, Sodium (CTP)
25 g 27-1006-03
Guanosine 5'-Triphosphate, Sodium (GTP)
1 g 27-1200-04 1 g 27-2000-04
For pricing information, visit www.gelifesciences.com/orderonline
Fluorescent Nucleotides For main product entry, see page 342.
Fluorescent Amidites For main product entry, see page 222.
ORDERING INFORMATION Product
Quantity Code Number
Cy5 Amidite
100 mg
27-1801-02
Cy5.5 Amidite
100 mg
27-1799-01
For pricing information, visit www.gelifesciences.com/orderonline
Radiolabeled dNTPs For main product entries, see Chapter 3.
Fluorescein Amidites
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 189 - CURRENCY: USD
High Purity Solution rNTPs
For main product entry, see page 222.
GE Healthcare
For more information, visit www.gelifesciences.com
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Nucleic Acid Sample Preparation / Protein Sample Preparation Nucleotides / Introduction to Trap Kits for Protein Sample Preparation Other Nucleotides For information concerning molecular weights, structures and extinction coefficients for representative nucleotides, see page 665.
ORDERING INFORMATION Product Diguanosine Triphosphate, Sodium [G(5')ppp(5')G] m7G(5')ppp(5')G, Sodium
ORDERING INFORMATION Product
Quantity Code Number
Miscellaneous Nucleotides
Quantity Code Number
Cap Analogue Nucleotides for mRNA Transcription 25 A250 units 5 A250 units
27-4643-01 27-4635-01
m7G(5')ppp(5')G, Sodium
25 A250 units
27-4635-02
m7G(5')ppp(5')G, Sodium
100 A250 units
27-4635-03
25 mg 27-7350-02
Coenzyme A and Derivatives
2'-Deoxycytidine 5'-O-(1-Thiotriphosphate), Sodium (dCTPaS) 5-Methyldeoxycytidine 5'-Triphosphate
25 mg 27-7360-02
Coenzyme A (CoA-SH), Free Acid, Chromatographically Purified
500 mg
28-3001-03
5 mg 27-4225-01
5-Methyldeoxycytidine 5'-Triphosphate 3'-O-Methylguanosine 5'-Triphosphate, Sodium
25 mg 27-4225-02 1 µmol 27-4675-01
n-Butyryl Coenzyme A, Lithium n-Butyryl Coenzyme A, Lithium
25 mg 100 mg
27-5820-02 27-5820-03
For pricing information, visit www.gelifesciences.com/orderonline
For pricing information, visit www.gelifesciences.com/orderonline GE Healthcare also offers bulk packaging and custom blends of nucleotides and modified nucleotides. Contact your local GE Healthcare representative for further information.
5
The Trap range of kits are designed to bring consistency, quality, and reliability to sample preparation for electrophoresis, liquid chromatography, and mass spectrometry. For an overview of available products for protein sample preparation, see Selection Guides on pages 151 and 152.
For more information, visit www.gelifesciences.com
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Due to the great diversity of protein sample types and origin, different treatments and conditions are required to extract and solubilize different types of protein samples. Some proteins are naturally found in complexes with membranes, nucleic acids, or other proteins; some proteins form various nonspecific aggregates; other proteins precipitate when removed from their normal environment. The effectiveness of solubilization depends on the choice of cell disruption method, protein concentration and dissolution method, choice of detergent, and composition of sample solution.
If any of these steps are not optimized for a particular sample and/or analysis, separations may be incomplete or distorted and information may be lost. Also, accurate quantitation of the final sample helps in determining the right protein load and increases the chances of visualizing low-abundance proteins.
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190
J High-quality kits for convenient sample preparation that give consistent and reliable results. J Effectively disrupt cells and tissues. J Cleanup samples from contaminants. J Desalting and buffer exchange of protein/carbohydrate/peptide samples. J Accurately quantitate protein for first-dimension isoelectric focusing. J Enrichment of specific proteins from a complex protein sample. J High purity chemicals and reagents for protein solubilization.
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DNA, RNA, Protein Sample Prep & DNA Amplification
Introduction to Trap Kits for Protein Sample Preparation
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2'-Deoxyadenosine 5'-O-(1-Thiotriphosphate), Sodium (dATPaS)
GE Healthcare
ch05.fm Page 191 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Lysis/Tissue Homogenization Sample Grinding Kit J Disrupts small tissue and cell samples for protein extraction. J Uses 1.5 ml microcentrifuge tubes, grinding resin, and disposable pestles. J Process up to 100 mg of sample per tube in about 10 min. Sample Grinding Kit is ready to use for disrupting small tissue and cell samples for protein extraction.
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Samples treated Sample weight Time Storage Shelf life Components Microcentrifuge tubes with grinding resin Pestles
50 Up to 100 mg 10 min Room temperature Unlimited
Product Sample Grinding Kit
Quantity Code Number 50 samples
80-6483-37
For pricing information, visit www.gelifesciences.com/orderonline
The kit consists of 1.5 ml microcentrifuge tubes, each containing a small quantity of abrasive grinding resin suspended in water, and disposable pestles for grinding. The tube is first centrifuged to pellet the resin and water is removed. Then extraction solution of choice and the sample are added to the tube, and the pestle is used to grind the sample. After centrifugation, cellular debris and grinding resin pellet firmly in the bottom of the conical tube, and the supernatant is easily pipetted out.
5
50 50
Schematic of the method used in Sample Grinding Kit. (a) Grinding resin pelleted in microcentrifuge tube (b) Sample and extraction solution added and sample disrupted by grinding with pestle. (c) Spin out cellular debris and resin. (d) Collect supernatant.
DIGE approved
Protease Inhibitor Mix J Protease inhibitor cocktail specifically developed for sample preparation in 2-D studies. J No EDTA is used, which allows optimal nuclease activity for removing nucleic acids from samples. J Contains a mixture of both competitive and noncompetitive protease inhibitors that inhibit serine, cysteine, and calpain proteases. Optionally, EDTA may be added to inhibit metalloproteases. J Optimized concentration for excellent inhibition of protease activities. J Effectively inhibits over 95% of protease activity.
ORDERING INFORMATION Product Protease Inhibitor Mix
1 ml
80-6501-23
For pricing information, visit www.gelifesciences.com/orderonline
Sample preparation often requires the inhibition of protease activity. GE Healthcare offers this unique combination of competitive and noncompetitive protease inhibitors, which protect proteins from proteolysis during purification from animal tissues, plant tissues, yeast, and bacteria.
DIGE approved
Nuclease Mix J Specifically developed for removal of nucleic acids in sample preparation for IEF/2-D electrophoresis applications in proteomic studies. J Compatible with Protease Inhibitor Mix. J Ready-to-use: Simply add the mix to your sample and incubate. J Does not contain EDTA, which is an inhibitor of nuclease.
GE Healthcare
Quantity Code Number
ORDERING INFORMATION Product Nuclease Mix
Quantity Code Number 0.5 ml
80-6501-42
For pricing information, visit www.gelifesciences.com/orderonline
Removal of nucleic acids is often required to avoid contamination and subsequent artifacts on 2-D gels. The Nuclease Mix offers an effective mix of DNase and RNase enzymes, as well as the necessary cofactors for optimal nuclease activity.
For more information, visit www.gelifesciences.com
DNA, RNA, Protein Sample Prep & DNA Amplification
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TECHNICAL SPECIFICATIONS
ORDERING INFORMATION
191
ch05.fm Page 192 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation High-Abundant Protein Depletion
DIGE approved
Albumin and IgG Removal Kit
Quantity Code Number
Albumin and IgG Removal Kit
10 samples
RPN6300
For pricing information, visit www.gelifesciences.com/orderonline
Schematic of the removal process. Optimal albumin and IgG binding (y 95% total protein) is achieved using a 15 µl human serum loading and will typically lead to recovery of between 150 to 220 µg of low-abundance proteins. The amount of protein recovered will vary with the protein content of the individual serum samples used.
TECHNICAL SPECIFICATIONS Samples per kit Sample volume Storage Components Albumin IgG removal affinity gel slurry 50/50 (v/v) Empty MicroSpin columns Microcentrifuge tubes and lids
10 samples of 15 µl human serum 10 to 15 µl 2°C to 8°C for affinity gel, room temperature for tubes 8.5 ml
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10 10
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192
The Albumin and IgG Removal Kit improves resolution of low-abundance proteins and increases the number of spots in the treated sample.
Product
PAGE: 192 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
Proteins in serum and other biological fluids are difficult to resolve by 2-D electrophoresis, largely due to the abundance of serum albumin and IgG. In human serum, albumin constitutes 50% to 70% of the total protein and IgG constitutes 10% to 25%. The presence of these proteins obscures other proteins in the gel and limits the amounts of proteins in the serum that can be resolved by 2-D electrophoresis. In addition, these proteins have wide pI and molecular weight ranges that further reduce resolution and mask other low-abundance proteins.
ORDERING INFORMATION
PAGE: 192 - CURRENCY: USD
J Innovative adsorption gel: selective and efficient removal of y 95% albumin and IgG from 10 to 15 µl of human serum samples prior to 2-D electrophoresis. J Versatile: simultaneously removes the two most abundant proteins, albumin and IgG, saving time and preventing protein loss. J Convenient format: easy to use with minimal handling steps. J Complete kit, includes everything you need for 10 sample preparations: easy setup and quick results. J Simple optimized protocol: provides accurate, consistent, and reproducible results.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 193 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Sample Concentration Vivaspin Sample Concentrators ORDERING INFORMATION
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Vivaspin enables up to 30-fold concentration of biological sample.
J One-step sample concentration in a single tube for minimal sample handling and reduced sample loss. J Patented dead-stop technology, which ensures that samples cannot be concentrated to dryness and enables direct concentrate recovery. J Vertical polyethersulfone membrane minimizes membrane blockage and tolerates high flow rates. J Easy, contact-free storage by reverse spinning the concentrate into the recovery cap (Vivaspin 2). J Compatible pH range is from pH 1 to 9. Vivaspin sample concentrators are designed for fast, nondenaturing concentration of biological samples by membrane ultrafiltration. Up to 30-fold concentration of the sample can be achieved with recovery of the target molecule typically exceeding 95%. The entire process is performed in a single tube with an upper compartment containing sample and lower compartment separated by a semipermeable membrane with a molecular weight cut-off (MWCO) selected by the user. Centrifugation is applied to force solvent through the membrane, leaving a more concentrated sample in the upper chamber. Vivaspin sample concentrators cater for sample volumes from 100 µl to 20 ml, with a range of molecular weight cutoff values from Mr 3 000 to 100 000. For maximum recovery, select a MWCO at least 50% smaller than the molecular size of the species of interest.
GE Healthcare
Quantity Code Number
Vivaspin 500 MWCO 3 000 Z
25
28-9322-18
Vivaspin 500 MWCO 5 000 Z Vivaspin 500 MWCO 10 000 Z
25 25
28-9322-23 28-9322-25
Vivaspin 500 MWCO 30 000 Z
25
28-9322-35
Vivaspin 500 MWCO 50 000 Z Vivaspin 500 MWCO 100 000 Z
25 25
28-9322-36 28-9322-37
Vivaspin 2 MWCO 3 000 Z Vivaspin 2 MWCO 5 000 Z
25 25
28-9322-40 28-9322-45
Vivaspin 2 MWCO 10000 Z Vivaspin 2 MWCO 30000 Z
25 25
28-9322-47 28-9322-48
Vivaspin 2 MWCO 50000 Z
25
28-9322-57
Vivaspin 2 MWCO 100000 Z Vivaspin 6 MWCO 3 000 Z
25 25
28-9322-58 28-9322-93
Vivaspin 6 MWCO 5 000 Z Vivaspin 6 MWCO 10000 Z
25 25
28-9322-94 28-9322-96
Vivaspin 6 MWCO 30000 Z Vivaspin 6 MWCO 50000 Z
25 25
28-9323-17 28-9323-18
Vivaspin 6 MWCO 100000 Z Vivaspin 20 MWCO 3 000 Z
25 12
28-9323-19 28-9323-58
Vivaspin 20 MWCO 5 000 Z
12
28-9323-59
Vivaspin 20 MWCO 10 000 Z Vivaspin 20 MWCO 30 000 Z
12 12
28-9323-60 28-9323-61
Vivaspin 20 MWCO 50 000 Z Vivaspin 20 MWCO 100 000 Z
12 12
28-9323-62 28-9323-63
For pricing information, visit www.gelifesciences.com/orderonline TECHNICAL SPECIFICATIONS Membrane Body Filtrate vessel Storage temperature
Concentrator capacity, swing bucket rotor Concentrator capacity, fixed angle rotor Length Width Active membrane area Hold-up volume of membrane Dead-stop volume
Polyethersulfone (PES) Polycarbonate Polycarbonate 4°C to 30°C Vivaspin 500 2 Do not use 3 ml
6 6 ml
20 20 ml
500 µl
2 ml
6 ml
14 ml
50 mm 11 mm 0.5 cm2 v 5 µl
126 mm 17 mm 1.2 cm2 v 10 µl
122 mm 17 mm 2.5 cm2 v 10 µl
116 mm 30 mm 6.0 cm2 v 20 µl
5 µl
8 µl
30 µl
50 µl
Related Products
Code Number
Disposable PD-10 Desalting Columns IMPROVED
17-0851-01
page 503
PD SpinTrap G-25 Z PD MultiTrap G-25 Z
28-9180-04 28-9180-06
page 205 page 206
PD MiniTrap G-25 Z
28-9180-07
page 207
PD MidiTrap G-25 Z PD MiniTrap G-10 Z
28-9180-08 28-9180-10
page 208 page 209
PD MidiTrap G-10 Z
28-9180-11
page 210
For more information, visit www.gelifesciences.com
Refer To
5
DNA, RNA, Protein Sample Prep & DNA Amplification
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Product
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Protein Sample Preparation Fractionation
DIGE approved
2-D Fractionation Kit ORDERING INFORMATION
J Proven technology resolves complex protein samples into six easily manageable fractions. J High sample recovery with no loss of protein. J Produces more abundant spots. J Enhances the visibility and detection of low-abundance proteins. J Can be scaled up for larger samples. J Compatible with other downstream separation techniques, such as 2-D electrophoresis.
80-6501-04
For pricing information, visit www.gelifesciences.com/orderonline TECHNICAL SPECIFICATIONS Samples per kit Storage
Fractionation makes it possible to isolate groups of proteins, or fractions from the total proteome. This allows for improved resolution when an individual fraction is analyzed, provides less crowded 2-D maps, simplifies analysis and interpretation, and increases the chances of discovering novel proteins of diagnostic or therapeutic interest. The 2-D Fractionation Kit simplifies analysis of complex protein mixtures by reducing the amount and number of protein species loaded into the gel matrix.
Shelf life Components Lysis buffer Fraction precipitant Precipitant Co-precipitant Wash buffer Solubilizer and Diluent
10 samples of 0.5 ml each Room temperature, some components need to be refrigerated after opening 1 yr 50 ml 15 ml 2 u 30 ml 2 u 30 ml 50 ml Makes 50 ml buffer
Tissue / Cells Lysis buffer Sonicate Spin Supernatant
Pellet
0.1 ml Fraction Precipitant, incubate, spin Pellet = Fraction I
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Rewash Insoluble fraction
Supernatant 0.1 ml Fraction Precipitant, incubate, spin
Pellet = Fraction II
Solubilize
2-D gel
Supernatant 0.2 ml Fraction Precipitant, incubate, spin
Pellet = Fraction III
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DNA, RNA, Protein Sample Prep & DNA Amplification
10 samples
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194
Quantity Code Number
2–D Fractionation Kit
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5
Product
Supernatant
Further additions of Fraction Precipitant or Precipitant: Pellets = Fractions IV, V, VI
(Wash VI), dialyze all in buffer of choice
2-D gels
Schematic of fractionation workflow using 2-D Fractionation Kit.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 195 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Protein Quantitation
DIGE approved
2-D Clean-Up Kit J Improves the quality of 2-D electrophoresis results by removing interfering contaminants. J Quantitative precipitation separates proteins from detergents, salts, lipids, phenolics, and nucleic acids. J Delivers quantitative yield in about 90 min.
PAGE: 195 - CURRENCY: USD
Quantity Code Number
2-D Clean-Up Kit
50 samples
80-6484-51
For pricing information, visit www.gelifesciences.com/orderonline TECHNICAL SPECIFICATIONS Samples per kit Sample volume Time Storage Shelf life Components: Precipitant Co-precipitant Wash buffer Wash additive
50 (fewer if sample y 100 µl) Up to 100 µl 1.5 h Room temperature 1 yr 15 ml 15 ml 50 ml 250 µl
The 2-D Clean-Up Kit procedure uses precipitant and co-precipitant in combination to quantitatively precipitate proteins. Precipitated proteins are pelleted by centrifugation and the precipitate is further washed to remove nonprotein contaminants. After a second centrifugation, the resultant pellet is resuspended into denaturing sample solution for first-dimension IEF. The procedure can be completed in 90 min and does not result in spot gain or loss. The kit contains sufficient reagents to process 50 samples of up to 100 µl each. The procedure can be scaled up for larger volumes or more dilute samples. 2-D Clean-Up Kit increases labeling efficiency and protein abundance when using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Please request application note Improving Sample Quality in 2-D Difference Gel Electrophoresis using Ettan 2-D Clean-Up Kit (code number 18-1164-86).
2-D Clean-Up Kit eliminates horizontal streaking caused by residual SDS. Sample: Rat liver extracted with 4% SDS, 40 mM Tris base. First dimension: Approximately 20 µg of rat liver protein, 7 cm Immobiline DryStrip pH 4–7, Ettan IPGphor IEF unit 17.5 kVh. Second dimension: SDS-PAGE (12.5%), SE 260 (8 u 9 cm gel). Stain: PlusOne Silver Staining Kit, Protein.
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Treatment of the sample with 2-D Clean-Up Kit can improve the quality of 2-D electrophoresis results, reducing streaking, background staining, and other artifacts due to interfering contaminants. The kit can make 2-D analysis possible for samples that are otherwise too dirty or too dilute.
Product
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 195 - CURRENCY: USD
2-D Clean-Up Kit is designed to prepare samples for 2-D electrophoresis that otherwise produce poor 2-D spot-maps due to high conductivity, high levels of interfering substances, or low protein concentration. The reagents quantitatively precipitate proteins while leaving interfering substances, such as detergents, salts, lipids, phenolics, and nucleic acids, in solution.
ORDERING INFORMATION
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Protein Sample Preparation Untagged Protein Enrichment NHS HP SpinTrap J Simple, small-scale enrichment of target proteins. J Antibody or capturing molecule covalently immobilized via free NH2 groups. J Simple coupling, carried out near neutral pH. J Fast and flexible protocol for the capture step, with elution conditions formatted for both electrophoresis and LC-MS analysis workflows. J Easy scale-up to HiTrap NHS-activated HP Columns. J The kit contains bulk media and empty SpinTrap columns to allow for batch coupling*.
Quantity Code Number 5 ml media plus 24 columns
28-9031-28
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
HiTrap NHS-activated HP Columns NHS HP SpinTrap Buffer Kit Z
Refer To page 473
28-9135-69
this page
TECHNICAL SPECIFICATIONS Group to be coupled Coupling conditions Matrix Medium Substitution Spacer arm Average particle size pH stability (ligand dependent) Max. sample loading volume Column material Storage Storage temperature
-NH2 pH 6.5 to 9 at 4°C to 25°C Highly cross-linked agarose 6% NHS Sepharose High Performance 10 µmol NHS/ml medium 6-aminocaproic acid, 10-atom 34 µm 3 to 12 600 µl Polypropylene barrel and polyethylene frits 100% isopropanol 4°C to 30°C
NHS HP SpinTrap Buffer Kit ORDERING INFORMATION Product
Quantity Code Number
NHS HP SpinTrap Buffer Kit Z
1 28-9135-69
For pricing information, visit www.gelifesciences.com/orderonline Note: HCl and urea (optional) are not included in the kit. Related Products
Code Number
Refer To
NHS HP SpinTrap
28-9031-28
this page
10x Binding/Washing Buffer (TBS) 10x Elution Buffer 5x Coupling Buffer 5x Blocking Buffer A 5x Blocking Buffer B NHS HP SpinTrap Buffer Kit for convenient use with NHS HP SpinTrap columns in immunoprecipitation.
J Pre-made buffers optimized for the affinity-based enrichment of a target protein/immunoprecipitation using NHS SpinTrap HP columns. J High quality buffers. J All buffer components defined. J Convenient format — stock solutions can be diluted directly into the provided bottles for storage. J Eliminates time-consuming buffer preparation, which allows fast, reproducible protein enrichment.
0.5 M Tris, 1.5 M NaCl, pH 7.5 1 M glycine-HCl, pH 2.9 0.75 M triethanolamine, 2.5 M NaCl, pH 8.3 2.5 M ethanolamine, 2.5 M NaCl, pH 8.3 0.5 M sodium acetate, 2.5 M NaCl, pH 4.0
The buffers supplied are sufficient for performing 24 reactions.
The buffers provided in the kit are optimized for standard affinity-based enrichment/immunoprecipitation according to the protocol provided with the NHS HP SpinTrap columns.
For more information, visit www.gelifesciences.com
GE Healthcare
PAGE: 196 - CURRENCY: USD
TECHNICAL SPECIFICATIONS
PAGE: 196 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
NHS HP SpinTrap
PAGE: 196 - CURRENCY: USD
196
* NHS HP SpinTrap columns are used with a standard microcentrifuge.
Product
PAGE: 196 - CURRENCY: USD
5
NHS HP SpinTrap includes empty SpinTrap columns and NHSactivated Sepharose High Performance medium in bulk to allow for batch coupling. Proteins of interest are captured and enriched via an antibody or other molecule (e.g., protein) with desired affinity coupled to the medium through the primary amine. NHS Sepharose High Performance Medium is delivered in 100% isopropanol to protect the active groups.
ORDERING INFORMATION
ch05.fm Page 197 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Streptavidin HP SpinTrap ORDERING INFORMATION Product Streptavidin HP SpinTrap
Quantity Code Number 16 columns
28-9031-30
For pricing information, visit www.gelifesciences.com/orderonline
Streptavidin HP SpinTrap can be used for protein enrichment, where a biotinylated antibody (or similar affinity molecule) is attached to the streptavidin and the protein of interest is enriched through the affinity interaction with the antibody. The columns can also be used for the direct enrichment of proteins that are biotinylated. By binding a specific biotinylated protein to the column, protein-protein interactions can also be investigated.
17-5112-01 17-5113-01
page 488 page 489
Refer To
Streptavidin HP MultiTrap
28-9031-31
page 198
Streptavidin HP SpinTrap Buffer Kit Z
28-9135-68
this page
TECHNICAL SPECIFICATIONS Ligand Matrix Medium Average particle size Binding capacity pH stability Medium volume Max. sample loading volume Column material Storage Storage temperature
Streptavidin Highly cross-linked agarose 6% Streptavidin Sepharose High Performance 34 µm y 300 nmol biotin/ml medium 6 mg biotinylated BSA/ml medium 4 to 9 (long term), 2 to 10.5 (short term) 100 µl 600 µl Polypropylene barrel and polyethylene frits 20% ethanol 4°C to 30°C
* Streptavidin HP SpinTrap columns are used with a standard microcentrifuge.
Streptavidin HP SpinTrap Buffer Kit
PAGE: 197 - CURRENCY: USD
PAGE: 197 - CURRENCY: USD
J For simple, small-scale protein enrichment through immobilized biotinylated biomolecules. J Fast and flexible protocol with elution conditions formatted for both electrophoresis and LC-MS analysis workflows. J Useful for exploiting either the strong interaction of biotin and streptavidin or the somewhat weaker interaction of 2-iminobiotin and streptavidin. J Each column is packed with Streptavidin Sepharose High Performance for reproducibility and high performance*. J Easy scale-up with HiTrap Streptavidin HP prepacked columns.
Code Number
HiTrap Streptavidin HP Streptavidin Sepharose High Performance
ORDERING INFORMATION Product
Quantity Code Number
Streptavidin HP SpinTrap Buffer Kit Z
1 28-9135-68
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
Refer To
Streptavidin HP SpinTrap
28-9031-30
this page
PAGE: 197 - CURRENCY: USD
TECHNICAL SPECIFICATIONS 10 x Binding/Washing Buffer (TBS) 10 x Elution Buffer Blocking Buffer
0.5 M Tris, 1.5 M NaCl, pH 7.5 1 M glycine-HCl, pH 2.9 2 mM biotin; 50 mM Tris, 0,15 M NaCl, pH 7.5
The Buffers supplied are sufficient for performing 16 reactions.
The Streptavidin HP SpinTrap Buffer Kit for convenient use with the Streptavidin HP SpinTrap columns.
The buffers provided in the kit are optimized for standard affinity-based enrichment/immunoprecipitation according to the protocol provided with Streptavidin HP SpinTrap columns.
J Pre-made buffers optimized for the affinity-based enrichment of a target protein/immunoprecipitation using Streptavidin HP SpinTrap columns. J High quality buffers. J All buffer components defined. J Convenient format—stock solutions can be diluted directly into the provided bottles for storage. J Eliminates time-consuming buffer preparation, which allows fast, reproducible protein enrichment.
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 197 - CURRENCY: USD
Streptavidin HP SpinTrap are prepacked, single-use spin columns for protein enrichment through immobilized biotinylated biomolecules.
Related Products
197
ch05.fm Page 198 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Streptavidin HP MultiTrap ORDERING INFORMATION Product
Quantity Code Number
Streptavidin HP MultiTrap
4 u 96-well plates
28-9031-31
Collection plate 500 µl V-bottom
5 u 96-well plates
28-4039-43
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
Streptavidin HP SpinTrap
28-9031-30
Refer To page 197
HiTrap Streptavidin HP
17-5112-01
page 488
Streptavidin Sepharose High Performance
17-5113-01
page 489
Streptavidin HP MultiTrap, prepacked 96-well filter plates for protein enrichment through immobilized biotinylated biomolecules.
Average particle size Binding capacity pH stability Multiwell format Well volume Medium volume Max. sample loading volume Multiwell plate material Storage Storage temperature Filter plate size
PAGE: 198 - CURRENCY: USD
Streptavidin HP MultiTrap can be used for protein enrichment, where a biotinylated antibody (or similar affinity molecule) is attached to the Streptavidin and the protein of interest is enriched through the affinity interaction with the antibody. Streptavidin HP MultiTrap can also be used for the direct enrichment of proteins that are biotinylated. By binding a specific biotinylated protein to the column, protein-protein interactions can also be investigated. Remember to order the collection plates for washing and elution of the enriched protein, see ordering information.
For more information, visit www.gelifesciences.com
PAGE: 198 - CURRENCY: USD
198
J Fast, reliable protein enrichment through immobilized biotinylated biomolecules in prepacked 96-well filter plates that are ready to use. J Fast and flexible protocol with elution conditions formatted for both electrophoresis and LC-MS analysis workflows. J Useful for exploiting either the strong interactions of biotin and streptavidin or the somewhat weaker interaction of 2-iminobiotin and streptavidin. J Each well is prepacked with Streptavidin Sepharose High Performance for reproducibility and high performance. J Easy scale-up to Streptavidin HP SpinTrap or HiTrap Streptavidin HP prepacked columns. J MultiTrap 96-well filter plates can be used with centrifugation or vacuum, manually or in automated robotic systems.
Streptavidin Highly cross-linked agarose 6% Streptavidin Sepharose High Performance 34 µm y 300 nmol biotin/ml medium 6 mg biotinylated BSA/ml medium 4 to 9 (long term), 2 to 10.5 (short term) 96 well 800 µl 50 µl 600 µl Polypropylene barrels and frits, polyethylene bottom 20% ethanol 4°C to 30°C 127.8 u 85.5 u 30.6 mm (according to ANSI/SBS 1-2004, 3-2004, and 4-2004 standards; ANSI = American National Standards Institute; SBS = Society for Biomolecular Screening)
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DNA, RNA, Protein Sample Prep & DNA Amplification
5
Ligand Matrix Medium
PAGE: 198 - CURRENCY: USD
TECHNICAL SPECIFICATIONS
GE Healthcare
ch05.fm Page 199 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Protein A HP SpinTrap ORDERING INFORMATION Product Protein A HP SpinTrap
Quantity Code Number 16 columns
28-9031-32
For pricing information, visit www.gelifesciences.com/orderonline Protein A HP SpinTrap allows enrichment of proteins from a variety of biological samples.
There is also a protocol for efficient, small-scale purification of monoclonal and polyclonal antibodies from serum and cell culture supernatants in less than 20 min. No pretreatment of the sample is required.
17-6002-35
Refer To page 460 page 203
28-9031-33
page 200
Protein A/G HP SpinTrap Buffer Kit NHS HP SpinTrap Buffer Kit Z
28-9135-67 28-9135-69
page 201 page 196
Ab SpinTrap Ab Buffer Kit
28-4083-47 28-9030-59
page 467 page 468
TECHNICAL SPECIFICATIONS Ligand Ligand coupling method Ligand density Matrix Medium Binding capacity Average particle size pH stability Medium volume Max. sample loading volume Column material Storage Storage temperature
Native protein A N-hydroxysuccinimide activation B 3 mg protein A/ml medium Highly cross-linked agarose 6% Protein A Sepharose High Performance y 1 mg human IgG well 34 µm 3 to 9 (long term), 2* to 9 (short term) 100 µl 600 µl Polypropylene barrel and polyethylene frits 20% ethanol 4°C to 30°C
* pH below 3 is sometimes required to elute strongly bound IgG species. However, protein ligands may hydrolyze at very low pH.
* Protein A HP SpinTrap columns are used with a standard microcentrifuge.
PAGE: 199 - CURRENCY: USD
GE Healthcare
Code Number
Protein A HP MultiTrap
PAGE: 199 - CURRENCY: USD
PAGE: 199 - CURRENCY: USD
Protein A Sepharose HP has high affinity for the Fc region of IgG from a variety of species. The two options in the protein enrichment protocol allow the attached antigen (protein of interest) to be eluted separately (cross-link protocol) or together with the antibody (classic protocol). There is also a protocol for efficient, small-scale purification of monoclonal and polyclonal antibodies from serum and cell culture supernatants in less than 20 min. No pretreatment of the sample is required.
HiTrap Protein A HP Columns Immunoprecipitation Starter Pack
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 199 - CURRENCY: USD
J For simple, small-scale antibody purification or enrichment of specific proteins in prepacked, single-use spin columns that are ready to use. J Classic and cross-link protocols provide flexibility. J Elution conditions formatted for both electrophoresis and LCMS analysis workflows. J Fast binding kinetics and high capacity provide high yield. J Prepacked* with Protein A Sepharose High Performance for coupling of antibodies of IgG subclasses. J Easy scale-up with HiTrap Protein A HP prepacked columns.
Related Products
199
ch05.fm Page 200 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Protein A HP MultiTrap ORDERING INFORMATION Product
Quantity Code Number
Protein A HP MultiTrap
4 u 96-well plates
28-9031-33
Collection plate 500 µl V-bottom
5 u 96-well plates
28-4039-43
For pricing information, visit www.gelifesciences.com/orderonline Related Products
28-9031-32 28-9030-59
page 199 page 468
TECHNICAL SPECIFICATIONS Ligand Ligand coupling method Ligand density Matrix Medium Binding capacity Average particle size pH stability Multiwell format Well volume Medium volume Max. sample loading volume Multiwell plate material Storage Storage temperature Filter plate size
Native protein A N-hydroxysuccinimide activation B 3 mg protein A/ml medium Highly cross-linked agarose 6% Protein A Sepharose High Perfomance y 1 mg human IgG/well 34 µm 3 to 9 (long term), 2* to 9 (short term) 96 well 800 µl 50 µl 600 µl Polypropylene barrels and frits, polyethylene bottom 20% ethanol 4°C to 30°C 127.8 u 85.5 u 30.6 mm (according to ANSI/SBS 1-2004, 3-2004, and 4-2004 standards; ANSI = American National Standards Institute; SBS = Society for Biomolecular Screening)
* pH below 3 is sometimes required to elute strongly bound IgG species. However, protein ligands may hydrolyze at very low pH.
For more information, visit www.gelifesciences.com
PAGE: 200 - CURRENCY: USD
There is also a protocol for efficient, small-scale purification of monoclonal and polyclonal antibodies from serum and cell culture supernatants in less than 20 min. No pretreatment of the sample is required.
PAGE: 200 - CURRENCY: USD
200
17-6002-35
Protein A HP SpinTrap Ab Buffer Kit
PAGE: 200 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
J Fast, reliable ease-of-use format for small-scale purification of antibodies or enrichment of proteins of interest in prepacked 96-well filter plates that are ready to use. J Flexible format, with protocols for classic elution and crosslink method. J Elution conditions formatted for both electrophoresis and LCMS analysis workflows. J Fast binding kinetics and high capacity provides high yield. J Easy scale-up with HiTrap Protein A HP prepacked columns. J Prepacked with Protein A Sepharose High Performance for coupling of antibodies of IgG subclasses. J MultiTrap 96-well filter plates can be used with centrifugation or vacuum, manually or in automated robotic systems. Protein A Sepharose High Performance has high affinity for the Fc region of IgG from a variety of species. The two options in the protein enrichment protocol allow the attached antigen (protein of interest) to be eluted separately (cross-link protocol) or together with the antibody (classic protocol). Remember to order the collection plates for washing and elution of the enriched protein.
Refer To page 460 page 203
PAGE: 200 - CURRENCY: USD
Protein A HP MultiTrap, prepacked 96-well filter plates for the enrichment of your protein of interest from small sample volumes.
Code Number
HiTrap Protein A HP Columns Immunoprecipitation Starter Pack
GE Healthcare
ch05.fm Page 201 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Protein A/G HP SpinTrap Buffer Kit ORDERING INFORMATION Product
Quantity Code Number
Protein A/G HP SpinTrap Buffer Kit
1 28-9135-67
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
Protein A HP SpinTrap
28-9031-32
page 199
Refer To
Protein G HP SpinTrap
28-9031-34
this page
TECHNICAL SPECIFICATIONS 10x Binding/Washing Buffer (TBS) Elution Buffer (Classic) 10x Elution Buffer (Crosslink) 10x Crosslink Solution A 10x Crosslink Solution B
0.5 M Tris, 1.5 M NaCl, pH 7.5 2.5% acetic acid 1 M glycine-HCl, pH 2.9 2 M triethanolamine, pH 8.9 1 M ethanolamine, pH 8.9
The buffers supplied are sufficient for performing 16 reactions.
PAGE: 201 - CURRENCY: USD PAGE: 201 - CURRENCY: USD PAGE: 201 - CURRENCY: USD
J Pre-made buffers optimized for the affinity-based enrichment of a target protein/immunoprecipitation using Protein A HP, Protein G HP, and Ab SpinTrap columns. J High quality buffers. J All buffer components listed and defined. J Convenient format — stock solutions can be diluted directly into the provided bottles for storage. J Eliminates time-consuming buffer preparation, which allows fast, reproducible protein enrichment.
The buffers provided in the kit are optimized for standard affinity-based enrichment/immunoprecipitation according to the protocol provided with Protein A HP SpinTrap, Protein G HP SpinTrap, and Ab SpinTrap columns.
Protein G HP SpinTrap ORDERING INFORMATION Product Protein G HP SpinTrap
Quantity Code Number 16 columns
28-9031-34
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
HiTrap Protein G HP Columns
Protein G HP SpinTrap is ideal for enrichment of proteins from a variety of biological samples.
J For simple, small-scale purification of antibodies or enrichment of specific proteins in prepacked spin columns that are ready to use. J Classic and cross-link protocols provide flexibility. J Elution conditions formatted for both electrophoresis and LCMS analysis workflows. J Fast binding kinetics and high capacity provides high yield. J Prepacked* with Protein G Sepharose High Performance for coupling of antibodies of IgG subclasses. J Easy scale-up with HiTrap Protein G HP prepacked columns. Protein G Sepharose HP has high affinity for the Fc region of IgG from a variety of species. The two options in the protein enrichment protocol allow the attached antigen to be eluted separately (cross-link protocol) or together with the antibody (classic protocol). There is also a protocol for efficient, small-scale purification of monoclonal and polyclonal antibodies from serum and cell culture supernatants in less than 20 min. No pretreatment of the sample is required.
Refer To page 465
Protein A/G HP SpinTrap Buffer Kit Immunoprecipitation Starter Pack
28-9135-67 17-6002-35
this page page 203
Ab SpinTrap Ab Buffer Kit
28-4083-47 28-9030-59
page 467 page 468
Protein G HP MultiTrap
28-9031-35
page 202
TECHNICAL SPECIFICATIONS Ligand Ligand coupling method Ligand density Matrix Medium Binding capacity Average particle size pH stability Medium volume Max. sample loading volume Column material Storage Storage temperature
Recombinant protein G lacking albumin-binding region N-hydroxysuccinimide activation B 2 mg protein G/ml medium Highly cross-linked agarose 6% Protein G Sepharose High Performance y 1 mg human IgG/column 34 µm 3 to 9 (long term), 2* to 9 (short term) 100 µl 600 µl Polypropylene barrel and polyethylene frits 20% ethanol 4°C to 30°C
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 201 - CURRENCY: USD
Protein A/G HP SpinTrap Buffer Kit for convenient use with Protein A HP SpinTrap or Protein G HP SpinTrap columns in immunoprecipitation
* pH below 3 is sometimes required to elute strongly bound IgG species. However, protein ligands may hydrolyze at very low pH.
* Protein G HP SpinTrap columns are used with a standard microcentrifuge.
GE Healthcare
For more information, visit www.gelifesciences.com
201
ch05.fm Page 202 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Protein G HP MultiTrap ORDERING INFORMATION Product
Quantity Code Number
Protein G HP MultiTrap
4 u 96-well plates
28-9031-35
Collection plate 500 µl V-bottom
5 u 96-well plates
28-4039-43
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
Refer To
HiTrap Protein G HP Columns Immunoprecipitation Starter Pack
17-6002-35
page 465 page 203
Ab SpinTrap Ab Buffer Kit
28-4083-47 28-9030-59
page 467 page 468
Protein G HP SpinTrap
28-9031-34
page 201
Ligand
Protein G Sepharose HP has high affinity for the Fc region of IgG from a variety of species. The two options in the protein enrichment protocol allow the attached antigen to be eluted separately (cross-link protocol) or together with the antibody (classic protocol).
Binding capacity Average particle size pH stability Multiwell format Well volume Medium volume Max. sample loading volume Multiwell plate material Storage Storage temperature Filter plate size
* pH below 3 is sometimes required to elute strongly bound IgG species. However, protein ligands may hydrolyze at very low pH.
PAGE: 202 - CURRENCY: USD
A protocol for efficient, small-scale purification of monoclonal and polyclonal antibodies from serum and cell culture supernatants in less than 20 min is also provided. No pretreatment of the sample is required. Remember to order collection plates for washing and elution of the enriched protein.
For more information, visit www.gelifesciences.com
PAGE: 202 - CURRENCY: USD
202
J Fast, reliable easy-to-use format for small-scale purification of antibodies or enrichment of proteins of interest in prepacked 96-well filter plates that are ready to use. J Flexible format, protocol for classical elution as well as crosslinking method. J Formatted for elution for electrophoresis or LC-MS analysis workflows. J Fast binding kinetics and high capacity provides high yield. J Easy scale-up with HiTrap Protein G HP prepacked columns. J Prepacked with Protein G Sepharose High Performance for coupling of antibodies of IgG subclasses. J MultiTrap 96-well filter plates can be used with centrifugation or vacuum, manually or in automated robotic systems.
Ligand coupling method Ligand density Matrix Medium
Recombinant protein G lacking albumin-binding region N-hydroxysuccinimide activation B 2 mg protein G/ml medium Highly cross-linked agarose 6% Protein G Sepharose High Performance y 0.5 mg human IgG/well 34 µm 3 to 9 (long term), 2* to 9 (short term) 96 well 800 µl 50 µl 600 µl Polypropylene barrels and frits, polyethylene bottom 20% ethanol 4°C to 30°C 127.8 u 85.5 u 30.6 mm (according to ANSI/SBS 1-2004, 3-2004, and 42004 standards; ANSI = American National Standards Institute; SBS = Society for Biomolecular Screening)
PAGE: 202 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
Protein G HP MultiTrap is designed for enrichment of proteins from a variety of biological samples.
PAGE: 202 - CURRENCY: USD
TECHNICAL SPECIFICATIONS
GE Healthcare
ch05.fm Page 203 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment Immunoprecipitation Starter Pack ORDERING INFORMATION Product
Pack Size
Immunoprecipitation Starter Pack*
2 u 2 ml
Code Number 17-6002-35
For pricing information, visit www.gelifesciences.com/orderonline
PAGE: 203 - CURRENCY: USD PAGE: 203 - CURRENCY: USD PAGE: 203 - CURRENCY: USD
Immunoprecipitation Starter Pack consists of 2 ml nProtein A Sepharose 4 Fast Flow and 2 ml Protein G Sepharose 4 Fast Flow with instructions for analytical separations of target antigens from crude cell lysates.
J Contains two media, nProtein A Sepharose Fast Flow and Protein G Sepharose Fast Flow, ideal for immunoprecipitation of a wide range of antibodies with different binding selectivities. J Step-by-step instructions guide you through the procedure. J Contains 2 ml of each medium, sufficient for approximately 80 immunoprecipitations. J High specificity and high binding capacity with minimal nonspecific adsorption and low ligand leakage.
Related Products
Code Number
Protein A HP SpinTrap
28-9031-32
page 199
Protein A HP MultiTrap Protein G HP SpinTrap
28-9031-33 28-9031-34
page 200 page 201
Protein G HP MultiTrap nProtein A Sepharose 4 Fast Flow
28-9031-35 17-5280-01
page 202 page 461
Protein G Sepharose 4 Fast Flow Protein A Sepharose CL-4B
17-0618-01 17-0780-01
page 469 page 461
ExcelGel SDS Homogeneous 7.5 ExcelGel SDS Gradient 8-18
80-1260-01 80-1255-53
page 295 page 295
ExcelGel SDS Buffer Strips (anode and cathode)
17-1342-01
page 295
Hybond ECL (6 u 8 cm) ECL Western Blotting System
RPN68D RPN2108
page 352 page 357
Handbooks Affinity Chromatography Handbook: Principles and Methods
18-1022-29
page 568
Antibody Purification Handbook
18-1037-46
page 568
TECHNICAL SPECIFICATIONS
Ligand
Ligand density Ligand coupling method Matrix Binding capacity pH stability Storage Storage temperature
nProtein A Sepharose 4 Fast Flow Native protein A
B 6 mg Protein A/ml Cyanogen bromide activation Highly cross-linked agarose, 4% y 30 mg human IgG/ml medium 3 to 9 (working) 20% ethanol 4°C to 8°C
Ab SpinTrap For main product entries, see page 467.
Ab Buffer Kit For main product entries, see page 468.
Protein A / Protein G For main product entry, see page 83.
GE Healthcare
Refer To
For more information, visit www.gelifesciences.com
Protein G Sepharose 4 Fast Flow Recombinant protein G lacking albuminbinding region B 2 mg Protein G/ml Cyanogen bromide activation Highly cross-linked agarose, 4% y 20 mg human IgG/ml medium 3 to 9 (working) 20% ethanol 4°C to 8°C
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 203 - CURRENCY: USD
* Includes: nProtein A Sepharose 4 Fast Flow, 2 ml, and Protein G Sepharose 4 Fast Flow, 2 ml.
203
ch05.fm Page 204 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Untagged Protein Enrichment / Tagged Protein Capture Phos SpinTrap Fe ORDERING INFORMATION Product
Quantity Code Number
Phos SpinTrap Fe Z
1 28-9298-81
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
IMAC Sepharose 6 Fast Flow HiTrap IMAC FF
17-0921-07 17-0921-02
page 493 page 493
Refer To
Mono Q 5/50 GL
17-5166-01
page 521
Mono S 5/50 GL
17-5168-01
page 521
Matrix Chromatography medium
J Highly selective enrichment of phosphorylated peptides makes it easy to identify low abundant phosphorylated proteins by mass spectrometry. J Robust and reproducible capture performance. J Simple to follow protocol—Easy to adapt and optimize for specific analyses. Phos SpinTrap Fe is a kit designed for the enrichment of microgram quantities of phosphopeptides from mainly tryptic digests of cell culture lysates, as well as somatic fluids and tissues. The kit consists of a 2.5 ml bulk medium with Fe Sepharose 6 Fast Flow and 24 empty SpinTrap columns. Fe Sepharose 6 Fast Flow is an IMAC Sepharose 6 Fast Flow medium that is pre-activated with iron. Phosphopeptide fragments in the sample bind specifically to iron on Fe Sepharose 6 Fast Flow in a SpinTrap column.
Average particle size Working temperature Maximum sample volume Volume of the eluted sample Storage temperature Storage solution
Column size Column material and filter
PAGE: 204 - CURRENCY: USD PAGE: 204 - CURRENCY: USD
204
Phos SpinTrap Fe is a kit designed for enrichment of phosphopeptide fragments from a enzyme-digested protein sample.
PAGE: 204 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
Highly cross-linked 6% spherical agarose Fe Sepharose 6 Fast Flow 50% medium slurry, 2.5 ±0.2 ml 90 µm Room temperature 0.6 ml ( when using 80 µl medium slurry 0.5 ml 4°C to 30°C 20% ethanol, 50 mM 2-(Nmorpholino)ethane sulfonic acid), pH 5.5 ±0.2 0.8 ml Polypropylene barrel and polyethylene frits (10 µm)
PAGE: 204 - CURRENCY: USD
TECHNICAL SPECIFICATIONS
Schematic presentation of capture and enrichment using Phos SpinTrap Fe.
Tagged Protein Expression, Purification, and Detection For main product entry, see page 398.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 205 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup Disposable PD-10 Desalting Columns IMPROVED J Reliable and reproducible cleanup of proteins and other large biomolecules (y 5000 Mr). J Wide field of applications, such as desalting, buffer exchange, and removal of low-molecular weight compounds. J High desalting capacity. J Flexible and improved protocols with ability to run gravity flow or centrifugation. J Preparation of the samples in the range of 1.0 to 2.5 ml for gravity and 1.75 to 2.5 ml with centrifugation. For main product entry, see page 503.
PD SpinTrap G-25
5
ORDERING INFORMATION Product
Quantity Code Number
PAGE: 205 - CURRENCY: USD PAGE: 205 - CURRENCY: USD PAGE: 205 - CURRENCY: USD PAGE: 205 - CURRENCY: USD
50 columns
28-9180-04
PD SpinTrap G-25 is a single-use column for cleanup of biomolecules with a molecular weight y 5000.
J Convenient and fast small-scale cleanup of proteins and other large biomolecules (y 5000 Mr) with the possibility to run multiple samples in parallel. J Range of applications such as desalting, buffer exchange, and removal of low-molecular weight compounds. J High desalting capacity. J Reproducible, high throughput, parallel cleanup of protein samples. J Sample preparation in the range of 70 to 130 µl without sample dilution. PD SpinTrap G-25 is a single-use microspin column that is designed for desalting and buffer exchange of biological sample using a standard microcentrifuge. The column is prepacked with 500 µl Sephadex G-25 Medium, and the column material is biocompatible polypropylene.
Related Products
Code Number
PD MultiTrap G-25 Z
28-9180-06
page 206
Refer To
PD MiniTrap G-25 Z
28-9180-07
page 207
PD MidiTrap G-25 Z Disposable PD-10 Desalting Columns IMPROVED
28-9180-08 17-0851-01
page 208 page 503
Handbook Gel Filtration: Principles and Methods
18-1022-18
page 568
TECHNICAL SPECIFICATIONS Medium Separation mechanism Particle size range Exclusion limit Sample volume Bed volume Recovery* Desalting capacity Chemical stability Working pH range Storage Column material
Sephadex G-25 Medium Gel filtration 85 to 260 µm Mr 5000 70 to 130 µl 500 µl 70% to 90% y 85% All commonly used buffers 2 to 13 4°C to 30°C Polypropylene barrel and polyethylene frits
* Biomolecule dependent
PD SpinTrap G-25 sample preparation with a microcentrifuge.
GE Healthcare
For more information, visit www.gelifesciences.com
DNA, RNA, Protein Sample Prep & DNA Amplification
PD SpinTrap G-25 Z
For pricing information, visit www.gelifesciences.com/orderonline
205
ch05.fm Page 206 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup PD MultiTrap G-25 ORDERING INFORMATION Product
Quantity Code Number
PD MultiTrap G-25 Z
4 x 96-well plates
28-9180-06
Collection plate 500 µl V-bottom
5 u 96-well plates
28-4039-43
For pricing information, visit www.gelifesciences.com/orderonline Code Number
PD SpinTrap G-25 Z
28-9180-04
page 205
Refer To
PD MiniTrap G-25 Z
28-9180-07
page 207
PD MidiTrap G-25 Z Disposable PD-10 Desalting Columns IMPROVED
28-9180-08 17-0851-01
page 208 page 503
Handbook Gel Filtration: Principles and Methods
18-1022-18
page 568
TECHNICAL SPECIFICATIONS
J Convenient and fast small-scale cleanup of proteins and other large biomolecules (y 5000 Mr) with high reproducibility well-to-well and plate-to-plate. J Range of applications such as desalting, buffer exchange, and removal of low-molecular weight compounds. J High desalting capacity. J Suitable for manual use as well as for automation together with robotic systems. J Sample preparation in the range of 70 to 130 µl without sample dilution.
* Biomolecule dependent
PAGE: 206 - CURRENCY: USD
PD MultiTrap G-25 is designed for high-throughput desalting and buffer exchange of multiple samples. The 96-well plates are prepacked with 500 µl Sephadex G-25 Medium/well for high reproducibility. The plate material is biocompatible polypropylene.
PAGE: 206 - CURRENCY: USD
206
PD MultiTrap G-25 is a prepacked 96-well plate for cleanup of biomolecules with a molecular weight y 5000.
Sephadex G-25 Medium Gel filtration 85 to 260 µm Mr 5000 70 to 130 µl 500 µl 800 µl 70% to 90% y 85% All commonly used buffers 2 to 13 4°C to 30°C Polypropylene barrel and polyethylene frits
PAGE: 206 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
Medium Separation mechanism Particle size range Exclusion limit Sample volume Medium volume Well volume Recovery* Desalting capacity Chemical stability Working pH range Storage Column material
PAGE: 206 - CURRENCY: USD
Related Products
Reproducible removal of NaCl from BSA on PD MultiTrap G-25. Average desalting capacity is 93% and well-to-well variation 1%.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 207 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup PD MiniTrap G-25 ORDERING INFORMATION Product
Quantity Code Number
PD MiniTrap G-25 Z
50 columns
28-9180-07
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
PD SpinTrap G-25 Z PD MultiTrap G-25 Z
28-9180-04 28-9180-06
page 205 page 206
Refer To
PD MidiTrap G-25 Z
28-9180-08
page 208
Disposable PD-10 Desalting Columns IMPROVED Handbook
17-0851-01
page 503
Gel Filtration: Principles and Methods
18-1022-18
page 568
TECHNICAL SPECIFICATIONS
PAGE: 207 - CURRENCY: USD
* Biomolecule dependent
PD MiniTrap G-25 is designed for convenient desalting and buffer exchange of biological samples. For increased flexibility, the product has two alternative application protocols, using either gravity or centrifugation. By using the gravity protocol, a simple cleanup of the sample is done with one or several columns in parallel without any need for a purification system. With the centrifugation protocol, the samples are run in parallel in a standard centrifuge with minimal dilution of the eluted sample. Four adapters are included in each product pack to enable easy centrifugation. Removal of NaCl from BSA using the gravity protocol. Sample recovery was 95% and the desalting capacity was y 99%.
PAGE: 207 - CURRENCY: USD
PAGE: 207 - CURRENCY: USD
J Reliable and reproducible cleanup of proteins and other large biomolecules (y 5000 Mr). J Range of applications such as desalting, buffer exchange, and removal of low-molecular weight compounds. J High desalting capacity. J Flexible protocols with ability to use gravity flow or centrifugation. J Sample preparation in the range of 0.1 to 0.5 ml for gravity and 0.2 to 0.5 ml with centrifugation.
Sephadex G-25 Medium Gel filtration 85 to 260 µm Mr 5000 0.1 to 0.5 ml 0.2 to 0.5 ml 5 ml 2.1 ml 70% to 95% y 90% All commonly used buffers 2 to 13 4°C to 30°C Polypropylene barrel and polyethylene frits
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 207 - CURRENCY: USD
PD MiniTrap G-25 is a prepacked column for cleanup of biomolecules with a molecular weight y 5000 in sample volumes up to 0.5 ml.
Medium Separation mechanism Particle size range Exclusion limit Sample volume (gravity) Sample volume (spin) Column volume Medium volume Recovery* Desalting capacity Chemical stability Working pH range Storage Column material
207
ch05.fm Page 208 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup PD MidiTrap G-25 ORDERING INFORMATION Product
Quantity Code Number
PD MidiTrap G-25 Z
50 columns
28-9180-08
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
PD SpinTrap G-25 Z PD MultiTrap G-25 Z
28-9180-04 28-9180-06
page 205 page 206
Refer To
PD MiniTrap G-25 Z
28-9180-07
page 207
Disposable PD-10 Desalting Columns IMPROVED Handbook
17-0851-01
page 503
Gel Filtration: Principles and Methods
18-1022-18
page 568
J Reliable and reproducible cleanup of proteins and other large biomolecules (y 5000 Mr). J Range of applications such as desalting, buffer exchange, and removal of low-molecular weight compounds. J High desalting capacity. J Flexible protocols with ability to use gravity flow or centrifugation. J Sample preparation in the range of 0.5 to 1.0 ml for gravity and 0.75 to 1.0 ml with centrifugation.
* Biomolecule dependent
PD MidiTrap G-25 is designed for convenient desalting and buffer exchange of biological samples and volumes up to 1.0 ml can be handled. Two alternative application protocols are available, gravity or centrifugation. By using the gravity protocol, a simple cleanup of the sample is done with one or several columns in parallel without any need for a purification system. With the centrifugation protocol, the samples are run in parallel in a standard centrifuge with minimal dilution of the eluted sample. Four adapters are included in each product pack to enable easy centrifugation.
PAGE: 208 - CURRENCY: USD PAGE: 208 - CURRENCY: USD
208
PD MidiTrap G-25 is a prepacked column for cleanup of biomolecules with a molecular weight y 5000 in sample volumes up to 1.0 ml.
Sephadex G-25 Medium Gel filtration 85 to 260 µm Mr 5000 0.5 to 1.0 ml 0.75 to 1.0 ml 8.5 ml 70% to 90% y 90% 3.45 ml All commonly used buffers 2 to 13 4°C to 30°C Polypropylene barrel and polyethylene frits
PAGE: 208 - CURRENCY: USD
DNA, RNA, Protein Sample Prep & DNA Amplification
5
Medium Separation mechanism Particle size range Exclusion limit Sample volume (gravity) Sample volume (spin) Column volume Recovery* Desalting capacity Medium volume Chemical stability Working pH range Storage Column material
PAGE: 208 - CURRENCY: USD
TECHNICAL SPECIFICATIONS
Removal of NaCl from [3H]-heparan sulfate (HS) on a PD MidiTrap G-25 column. The fractions between the arrows contained 87% of the total recovery of [3H]-HS. The salt concentration was 20 mM after cleanup, corresponding to y 98% of the salt removal.
For more information, visit www.gelifesciences.com
GE Healthcare
ch05.fm Page 209 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup PD MiniTrap G-10 ORDERING INFORMATION Product
Quantity Code Number
PD MiniTrap G-10 Z
50 columns
28-9180-10
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Code Number
PD MidiTrap G-10 Z PD SpinTrap G-25 Z
28-9180-11 28-9180-04
page 210 page 205
Refer To
PD MultiTrap G-25 Z
28-9180-06
page 206
PD MiniTrap G-25 Z PD MidiTrap G-25 Z
28-9180-07 28-9180-08
page 207 page 208
Disposable PD-10 Desalting Columns IMPROVED Handbook
17-0851-01
page 503
Gel Filtration: Principles and Methods
18-1022-18
page 568
TECHNICAL SPECIFICATIONS
PD MiniTrap G-10 gravity columns give fast and simple desalting and buffer exchange without any need for a purification system. PD MiniTrap G-10 is a member of the Trap platform, which addresses the need for flexible, small-scale preparation of proteins and peptides before analysis.
PAGE: 209 - CURRENCY: USD
PAGE: 209 - CURRENCY: USD
PAGE: 209 - CURRENCY: USD
J Easy cleanup of peptides, small proteins, or carbohydrates larger than Mr 700, before downstream analysis such as liquid chromatography, electrophoresis, and MS. J Efficient removal of contaminants such as salts, dyes, and radioactive labels. J High desalting capacity. J Preparation of samples in the volume range 100 to 300 µl.
Sephadex G-10, cross-linked dextran Gel filtration 55 to 165 µl 700 Mr 100 to 300 µl 2.1 ml 5 ml All commonly used buffers 2 to 13 4°C to 30°C 20% ethanol Polypropylene barrel and polyethylene frits
Removal of NaCl from a [3H]-heparan sulfate octamer solution on a PD MiniTrap G-10 column. The fractions were analyzed with regard to conductivity and radioactive content. The oligosaccharide yield was approximately 95%, and the desalting capacity was 94%.
GE Healthcare
For more information, visit www.gelifesciences.com
5
DNA, RNA, Protein Sample Prep & DNA Amplification
PAGE: 209 - CURRENCY: USD
PD MiniTrap G-10 are prepacked, single-use columns for buffer exchange or cleanup of biological samples e.g., small proteins, peptides, and oligosaccharides.
Matrix Separation mechanism Average bead size Exclusion limit Sample volume Bed volume Column volume Chemical stability Working pH range Storage temperature Storage solution Column material
209
ch05.fm Page 210 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup PD MidiTrap G-10 ORDERING INFORMATION Product
Quantity Code Number
PD MidiTrap G-10 Z
50 columns
28-9180-11
For pricing information, visit www.gelifesciences.com/orderonline Code Number
PD MiniTrap G-10 Z PD SpinTrap G-25 Z
28-9180-10 28-9180-04
page 209 page 205
Refer To
PD MultiTrap G-25 Z
28-9180-06
page 206
PD MiniTrap G-25 Z PD MidiTrap G-25 Z
28-9180-07 28-9180-08
page 207 page 208
Disposable PD-10 Desalting Columns IMPROVED Handbook
17-0851-01
page 503
Gel Filtration: Principles and Methods
18-1022-18
page 568
TECHNICAL SPECIFICATIONS
J Easy cleanup of peptides, small proteins, or carbohydrates larger than Mr 700, before downstream analysis such as liquid chromatography, electrophoresis, and MS. J Efficient removal of contaminants such as salts, dyes, and radioactive labels. J High desalting capacity. J Preparation of samples in the volume range 400 to 1000 µl. PD MidiTrap G-10 gravity columns give fast and simple desalting and buffer exchange without any need for a purification system.
Sephadex G-10, cross-linked dextran Gel filtration 55 to 165µl 700 Mr 400 to 1000 µl 5.3 ml 8.5 ml All commonly used buffers 2 to 13 4°C to 30°C 20% ethanol Polypropylene barrel and polyethylene frits
Spin Adapters for Gravity Columns ORDERING INFORMATION Product
Quantity Code Number 10
28-9232-43
MidiSpin Adapter Z PD-10 Spin Adapter Z
10 10
28-9232-44 28-9232-45
For pricing information, visit www.gelifesciences.com/orderonline
J Convenient adapters, enable spinning of all gravity columns.
Related Products
Code Number
PD MiniTrap G-25 Z PD MidiTrap G-25 Z
28-9180-07 28-9180-08
page 207 page 208
Refer To
Disposable PD-10 Desalting Columns IMPROVED
17-0851-01
page 503
Adapters are polypropylene rings designed for use with the gravity columns PD MiniTrap G-25, PD MidiTrap G-25, and PD-10 Desalting Columns when the spin protocol is used. MiniSpin Adapter fits PD MiniTrap G-25 columns, MidiSpin Adapter fits PD MidiTrap G-25 columns, and PD-10 Spin Adapter fits PD-10 Desalting Columns.
Scaling up Desalting, see HiTrap and HiPrep Desalting Columns For main product entries, see Chapter 11.
For more information, visit www.gelifesciences.com
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MiniSpin Adapter Z
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PD MidiTrap G-10 are prepacked, single-use columns for buffer exchange or cleanup of biological samples e.g., small proteins, peptides and oligosaccharides.
Matrix Separation mechanism Average bead size Exclusion limit Sample volume Bed volume Column volume Chemical stability Working pH range Storage temperature Storage solution Column material
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DNA, RNA, Protein Sample Prep & DNA Amplification
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Related Products
ch05.fm Page 211 Tuesday, June 17, 2008 11:47 PM
Protein Sample Preparation Desalting/Buffer Exchange/Cleanup Mini Dialysis Kit J Designed for efficient dialysis of small sample volumes. J Uses disposable dialysis tubes with dialysis membrane incorporated into caps. J Conical tube bottom maximizes sample recovery. Dialyzing small samples can be difficult and inefficient when the sample must be transferred into and out of dialysis bags and centrifuge tubes. The disposable tubes in Mini Dialysis Kit offer a simple solution to the handling problems of small volume dialysis. The dialysis tubes in the kit each have a cap that incorporates a small disk of dialysis membrane. Samples are easily pipetted into and removed from the conical bottom of the tube. The capped tube is inserted into a float and then, with the cap and membrane down, placed in a stirred beaker containing the desired dialysis solution.
ORDERING INFORMATION Product
Quantity Code Number
Mini Dialysis Kit, 1 kDa cut-off, 250 µl
1 80-6483-75
Mini Dialysis Kit, 1 kDa cut-off, 2 ml Mini Dialysis Kit, 8 kDa cut-off, 250 µl
1 80-6483-94 1 80-6484-13
Mini Dialysis Kit, 8 kDa cut-off, 2 ml
1 80-6484-32
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Following dialysis, the tube is centrifuged briefly. This brings the entire contents of the tube back to the conical bottom for maximum recovery. The dialyzed sample can be stored in the same tube by simply replacing the dialysis cap with a standard cap.
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1 kDa, 250 µl 1 kDa, 2 ml 8 kDa, 250 µl 8 kDa, 2 ml A few hours to overnight 4°C to 8°C 1 yr 50 6 50
Schematic of the method used in Mini Dialysis Kit. (a) Cap with dialysis membrane, conical inner sample tube; (b) Introduce sample, screw on cap, slide tube into float; (c) Invert and dialyze while stirring; (d) Spin briefly to collect sample.
*MW cut-off is nominal
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MW cut-off* and maximum volume MW cut-off* and maximum volume MW cut-off* and maximum volume MW cut-off* and maximum volume Time Storage Shelf life Components Tubes with dialysis caps Floats Caps, standard
GE Healthcare
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DNA, RNA, Protein Sample Prep & DNA Amplification
TECHNICAL SPECIFICATIONS
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Protein Sample Preparation Desalting/Buffer Exchange/Cleanup SDS-PAGE Clean-Up Kit J Prepares samples for SDS-PAGE that are otherwise difficult to analyze because of salts or low protein concentrations. J Quantitatively precipitate proteins while leaving interfering substances in solution. J Delivers quantitative yield in less than 2 h.
ORDERING INFORMATION Product SDS-PAGE Clean-Up Kit
Quantity Code Number 50 samples
80-6484-70
For pricing information, visit www.gelifesciences.com/orderonline
SDS-PAGE Clean-Up Kit is designed to prepare samples for SDS-PAGE that are frequently difficult to analyze due to the presence of salts or low protein concentration.
TECHNICAL SPECIFICATIONS Samples per kit Sample volume Time Storage Shelf life Components Precipitant Co-precipitant Wash buffer Wash additive Buffer I Buffer II Sample buffer
50 (fewer if sample y 100 µl) 1 to 100 µl 2h Room temperature 1 yr 15 ml 15 ml 50 ml 250 µl 2 ml 500 µl 2.5 ml
Comparison of SDS-PAGE Clean-Up Kit with ethanol precipitation (a) Urinary protein precipitated with 10 volumes of ethanol. (b) Urinary protein precipitated with SDS-PAGE Clean-Up Kit. (c) Cerebrospinal fluid protein precipitated with SDS-Page clean-up Kit. (d) Cerebrospinal fluid protein precipitated with 10 volumes of ethanol. Gel: 8 x 9 cm, 12.5% acrylamide, 0.1% SDS, run on SE 260. Stain: Coomassie R250.
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DNA, RNA, Protein Sample Prep & DNA Amplification
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SDS-PAGE Clean-Up Kit uses a combination of a precipitant and co-precipitant to quantitatively precipitate the sample proteins while leaving interfering substances in solution. Detergents, salts, lipids, phenolics, and nucleic acids remain soluble while the proteins are pelleted by centrifugation. The precipitate is washed further to remove nonprotein contaminants and centrifuged again. The resultant pellet is resuspended, mixed with SDS-PAGE sample buffer, and heated. The sample is then ready for SDS-PAGE. The procedure can be completed in under 2 h with quantitative yield.
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GE Healthcare
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Protein Sample Preparation / Instrumentation for Purification and Desalting/Buffer Exchange/Cleanup
DIGE approved
2-D Quant Kit J Accurately determine protein concentration in the presence of 2% SDS, 1% DTT, 8 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte, and 2% IPG Buffer. J Quantitatively precipitates proteins while leaving interfering substances behind. J Linear response in the range of 0 to 50 µg protein, with recommended sample volumes of 1 to 50 µl.
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Common spectrophotometric methods of quantitating protein rely either on Coomassie dye binding or protein-catalyzed reduction of cupric (Cu2+) ion to cuprous (Cu+) ion. Dye-binding assays cannot be used in the presence of any reagent that also binds the dye. This includes carrier ampholytes and detergents such as PlusOne CHAPS, SDS, and Triton X-100. Assays that depend on the reduction of cupric ion cannot be used in the presence of reductants such as DTT, or in the presence of reagents that form complexes with cupric ion, such as thiourea or EDTA.
Product
Quantity Code Number
2-D Quant Kit
500 assays
80-6483-56
For pricing information, visit www.gelifesciences.com/orderonline Related Products
Refer To
Ultrospec 3100 pro UV/Visible Spectrophotometer
page 641
Reagents tested for compatibility with the 2-D Quant Kit Compound tested
Concentration
SDS CHAPS Triton X-100 Pharmalyte pH 3–10 IPG Buffer pH 3–10 NL Tris EDTA DTT 2-Mercaptoethanol Urea Thiorea Glycerol
2% (w/v) 4% (w/v) 1% (w/v) 2% (v/v) 2% (v/v) 50 mM 10 mM 1% (65 mM) 2% (v/v) 8M 2M 30% (w/v)
The 2-D Quant Kit procedure works by quantitatively precipitating proteins while leaving interfering substances behind. The assay is based on the specific binding of copper ions to protein. Precipitated proteins are resuspended in a copper-containing solution and unbound copper is measured with a colorimetric agent. The absorbance at 480 nm is inversely related to the protein concentration. The assay has a linear response to protein in the range of 0 to 50 µg and recommended sample volume is 1 to 50 µl. TECHNICAL SPECIFICATIONS Assays per kit Assay wavelength Linear range Sample volume Assay time Storage Shelf life Components: Precipitant Co-precipitant Copper solution Color reagent A Color reagent B BSA standard
500 480 nm 0 to 50 µg Up to 50 µl 1h 4°C to 8°C 1 yr 250 ml 250 ml 50 ml 2 u 250 ml 5 ml 5 ml (2 mg/ml)
2-D Quant Kit protein assay is resistant to interference from sample solution components.
Chromatography Systems & Accessories For main product entries, – ÄKTAdesign Systems, see page 570.
Spectrophotometry For main product entries,
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DNA, RNA, Protein Sample Prep & DNA Amplification
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2-D Quant Kit is designed for the accurate determination of protein concentration in samples prepared for electrophoresis techniques such as 2-D electrophoresis, SDS-PAGE, or IEF.
ORDERING INFORMATION
– Multi-Use Spectrophotometers, see pages 640 – 643.
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