antibody mediated HS

ultra low e.coli DNA

low foam

qscript

Quanta Core Technologies

Quanta BioSciences PCR Technologies

ENABLING PCR

Quanta BioSciences supplies superior quality conventional RT and PCR reagents for sophisticated applications in life science and drug discovery laboratories around the world. PerfeCting PCR is the goal Quanta BioSciences has enthusiastically strived for in designing a real time PCR and cDNA synthesis reagent portfolio that defines the standard for reproducibility, specificity and sensitivity.

Quanta Core Technologies ENABLING

antibody mediated HS

PCR

ultra low e.coli DNA

Legacy of Innovation Founding members of Quanta BioSciences have a legacy of leading the development of pioneering reagents including Invitrogen’s SuperScript® 1-Step RT-PCR Kits, Platinum® Taq, and Bio-Rad’s iScript™, and iQ™ /iTaq™ SuperMixes. Platinum® Pfx DNA Polymerase7

1999 SuperScript® 5’-RACE System1 SuperScript® 3’-RACE System2

Platinum® Taq High Fidelity5 ThermoScript™ RT-PCR Systems6

1992

1998 SuperScript® II One-Step RT-PCR Systems3 Platinum® Taq DNA Polymerase4 Platinum® Taq PCR SuperMixes

SuperScript First Strand Synthesis System for RT-PCR ThermoScript™ One-Step RT-PCR System9 Platinum® Taq PCRx DNA Polymerase10 Platinum® qPCR SuperMix-UDG ®

2000

8

iScriptTM cDNA Synthesis Kit iQTM SYBR® Green SuperMix iQTM Supermix iScriptTM One step RT PCR Kit qScriptTM cDNA SuperMix PerfeCTa® SuperMixes PerfeCTa® FastMixes

2002-2007

AccuMeltTM HRM qScript microRNA System PerfeCTa® ToughMixes qScriptTM XLT Kits GelTrackTM SuperMixes AccuStartTM II Taq DNA polymerase

2007-2012

1997 1 990

2015 1. Schuster, D., G. Buchman, and A. Rashtchian. (1992) A simple and efficient method for amplification of cDNA ends using 5’ RACE. Focus 14: 46-52. 2. Buchman, G.W. and A. Rashtchian. (1992) PCR amplification of nucleic acid sequences using the 3’ RACE system and direct cloning of the amplified products. Focus 14: 2-5. 3. Lee, E.H., Sitaraman, K., Schuster, D. and Rashtchian, A. (1997) A highly sensitive method for one-step amplification of RNA by polymerase chain reaction. Focus 19: 39-42 4. Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Improved specificity and yield with Platinum Taq DNA Polymerase. Focus 19: 46-47. 5. Schuster, D.M., Darfler, M., Lee, J.E., and Rashtchian, A. (1998) Improved sensitivity and specificity of RT-PCR. Focus 20: 33-34. 6. Schwabe, W., Lee, J.E., Xu, R.H., Sitaraman, K., Smith, M., Potter, R.J., Rosenthal, K., Rashtchian, A., and Gerard, G.F. (1998) ThermoScript™ RT, A new avian reverse transcriptase for high-temperature cDNA synthesis to improve RT-PCR. Focus 20: 30-33 7. Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999) Platinum Pfx DNA Polymerase for high fidelity PCR. Focus 21: 46 8. Thiel, V., Rashtchian, A., Herold, J., Schuster, D.M., Guan, N., and Siddell, S.G. (1997) Effective amplification of 20-kb DNA by reverse transcription PCR. Anal Biochem. 252(1):62-70. 9. Xu, R.H., Schuster, D.M., Lee, J.E., Smith, M., Potter, J., Dhariwal, G., Rosenthal, K., Nathan, M., Gerard, G.F., and Rashtchian, A. (2000) One-step analysis and quantification of RNA by RT-PCR using high-temperature reverse transcription. Focus 22: 3-5. 10. Borman, J., Schuster, D., Li, W., Jessee, J., and Rashtchian, A. (2000) PCR from problematic template. Focus 22: 10-11

CO N T E N T S Quanta BioSciences............................................................... Pg. 3 Extracta™ Genomic DNA Preparation Kits......................... Pg. 4 cDNA Synthesis...................................................................... Pg. 6 qScript XLT One-Step RT-PCR Kit......................................... Pg. 8 qScript XLT One-Step ToughMixes.................................... Pg. 10 AccuStart™ PCR.................................................................... Pg. 12 AccuStart II Mouse Genotyping........................................ Pg. 13 AccuStart II PCR ToughMix................................................. Pg. 14 AccuStart Genotyping ToughMix™.................................... Pg. 16

www.quantabio.com

 

AccuMelt™ HRM—High Resolution Melt Analysis SuperMix...................................................... Pg. 18 PerfeCta™ PreAmp SuperMix.............................................Pg. 20 qPCR SuperMixes and FastMixes™— SYBR® Green.........................................................................Pg. 22 PerfeCta FastMix II..............................................................Pg. 24 PerfeCta qPCR ToughMix....................................................Pg. 26 PerfeCta MultiPlex qPCR ToughMix..................................Pg. 28 qScript microRNA Quantification System........................Pg. 30 PerfeCta NGS Quantification Kits.....................................Pg. 32

Quanta BioSciences

3

Extracta Genomic DNA Preparation Kits DNA EXTRACTION MADE EASY

QUANTA’s Extracta sample preparation system can be scaled to process various amounts of solid tissue in a simple and fast single-tube procedure. DNA may be purified from many tissue types and is suitable for use in sensitive downstream applications such as PCR, qPCR and High Resolution Melt Analysis.

FEATURES AND BENEFITS

EXTRACTA TECHNOLOGY

■

Simple, fast protocol

■

Compatible with laboratory automation

■

■

■

DNA is suitable for qPCR, High Resolution Melt Analysis, endpoint PCR and other applications Works with wide range of tissue including hair, buccal swabs and mouse tails Optional 10 minute protocol for highthroughput applications

Extracta DNA Prep for PCR is a two-component reagent kit for rapid extraction of PCR-ready genomic DNA from mammalian tissues. Tissues are processed in 35 minutes with little hands-on time. Compared to a conventional proteinase K process this is significantly faster with fewer handling steps (Figure 1). The genomic DNA is suitable for sensitive downstream PCR applications including end-point PCR, High Resolution Melt Analysis (HRM) and quantitative real-time PCR (qPCR) without requiring any additional clean-up (Figures 2 & 3). In addition, the extracted DNA may be used in multiplexed PCR applications such as transgene or knock-out analyses. Tissue extractions can be done in tubes, plates or deep-well blocks to allow for adaptation to workflow and automation on liquid-handling workstations.

Tissue Sample Sizes and Extraction Reagent Volumes Sample

Size

Volume Comments

Mouse tails

0.2 to 0.5 cm

75 µL

Fresh or frozen tail samples can be used. Tail samples should be less than 0.5 cm.

Mouse ear punches

1 to 2 mm

50 µL

Ensure that the ear punches are completely submerged in Extraction Reagent.

Animal tissues

2 to 10 mg

100 µL

Tissue samples should be small and completely submerged in Extraction Reagent.

Buccal swabs 1 buccal brush 250 µL or swab

Collect cheek cells using a buccal brush or swab and place into Extraction Reagent in a 1.5 mL microcentrifuge tube. Twirl the brush or swab in the Extraction Reagent and carefully press and rotate the brush or swab against the side of the tube while removing it from the solution to ensure that most of the liquid remains in the tube.

Hair

1-3 hairs with root

75 µL

Trim hair shaft to 0.5 cm leaving the root intact and place root end down into Extraction Reagent.

Saliva

10-20 µL

100 µL

Mix samples well in Extraction Reagent.

ORDERING INFORMATION

4

PRODUCT

Quanta Cat. No.

Extracta DNA Prep

95091-025 25mL 95091-250 250mL

Pack Size

Extracta Genomic DNA Preparation Kits

ProtocolSPEED Speed and Yield Comparison PROTOCOL AND YIELD COMPARISON Figure 1

Extracta

Standard Protocol

35 min

>200 min

Add tissue to Extraction Reagent

Add PK to Buffer

DNA Yield Comparison

Add tissue to PK Digestion Buffer

Incubate 30 min at 95°C

Incubate > 3 hrs at 55°C

Add Stabilization Buffer

Multiplex Genotyping

Phenol Chloroform Extract

PCR

Remove supernatent to a new tube Ethanol Precipitate PCR

Similar yields of DNA were obtained from 2 mm mouse tail snips extracted in 150 L of Extracta DNA Prep for PCR - Tissue or by a standard protocol consisting of proteinase K digestion, phenol chloroform extraction and ethanol precipitation. 1.5 L of each extract was used in 20 L PCR amplification of mouse brain-derived neurotropic factor using PerfeCTa SYBR Green FastMix. PCR data was obtained in triplicate and converted to number of copies of target detected using a standard curve generated from purified mouse genomic DNA.

HUMAN TISSUES Extracta extracts produce similar TaqMan and SYBR Green results for human IL1

Figure 2

PerfeCTa qPCR SuperMix with a TaqMan probe and PerfeCTa SYBR Green FastMix

Multiplex Genotyping Figure 3

1

Melting Analysis

100

2

3

4

90 80

- dI / dT (%)

70 60 50

5

6

150 L mouse tail extracts 2 L of Extracta extract in 20 L PCR PerfeCTa SYBR Green FastMix

XPC XPC + p53 p53

40 30 20 10

p53

0 -10

XPC 80

81

82

83

84 85 86 Temperature [°C]

87

88

89

90

Threshold: 100%

Extracta Genomic DNA Preparation Kits

5

cDNA Synthesis QSCRIPT CDNA SYNTHESIS KITS FOR EXCEPTIONAL CDNA SYNTHESIS AND ACCURATE REPRESENTATION FROM LESS STARTING MATERIAL

qScript cDNA Synthesis Kits set a new standard for reproducibility, specificity, speed and sensitivity in quantitative and conventional RT-PCR. Key to the performance of qScript cDNA Synthesis Kits is our proprietary qScript Reverse Transcriptase, a mixture of an engineered M-MLV reverse transcriptase derivative and ribonuclease inhibitor protein, optimized for sensitive and reliable cDNA synthesis over a broad dynamic range of input RNA. Our cDNA synthesis reagents are provided in a variety of supermix and kit formats to fit your specific application.

FEATURES AND BENEFITS cDNA SuperMix—One tube, less variability, ideal for high throughput applications ■

Unbiased Representation—For confidence that your RNA is represented accurately in the resulting cDNA ■

Broad Dynamic Range—More reliably attain useful data from your precious samples ■

■

Flexible Priming—Choose the ideal priming method for your needs

qScript cDNA SuperMix Just add RNA and go! qScript cDNA SuperMix is the first commercially available true cDNA SuperMix. qScript cDNA SuperMix is an easy-to-use format for 2-step RT-PCR. This one-tube SuperMix contains all components for cDNA synthesis including buffer, dNTPs, MgCl2, primers, RNase inhibitor, qScript Reverse Transcriptase and stabilizers­–all you need to add is RNA. Complete cDNA synthesis is achieved in only 40 minutes (See Figure 1). Consistency and reproducibility are achieved over a broad dynamic range regardless of RNA input (See Figure 2). In addition, qScript cDNA SuperMix offers superior sensitivity and more accurate representation of low abundance genes compared to other products (See Figure 3). The SuperMix format eliminates multiple component additions providing exceptional reproducibility and precision. qScript cDNA SuperMix is available in both single tube and 96-well plate formats.

6

cDNA Synthesis

qScript cDNA Synthesis Kit qScript cDNA Synthesis Kit* is a sensitive and easy-to-use kit for RNA quantification using two-step RT-PCR. This kit contains an optimized blend of random and oligo(dT) primers for robust, consistent and unbiased first-strand synthesis over a broad range of RNA template concentrations. The novel qScript Reaction Mix (5X) contains all required reagents for cDNA synthesis, except for qScript Reverse Transcriptase and RNA. qScript Reverse Transcriptase is provided in a separate tube. *This kit is intended for use by researchers using Bio-Rad’s iScript cDNA Synthesis Kit (previously developed and manufactured by Quanta) for ongoing studies or established Standard Operating Procedures that cannot be readily switched to qScript cDNA SuperMix.

qScript Flex cDNA Kit The qScript Flex cDNA Synthesis Kit is an easy-to-use format for first strand cDNA synthesis that supports multiple RNA priming strategies. The qScript Flex Reaction Mix (5X) is optimized for use with oligo dT, random or gene-specific primers in any combination. A proprietary cDNA-priming enhancer that improves sensitivity is provided pre-mixed with random and oligo dT primers, and in a separate tube for use with gene-specific primers (user provided). qScript Reverse Transcriptase is provided in a separate tube.

QSCRIPT CDNA SYNTHESIS: PERFORMANCE DATA Conventional cDNA Synthesis Kit Protocol 1 hour 42 minutes

Mix RNA + water with 4 µl of cDNA SuperMix Final vol = 20 µl

Combine RNA template, dNTPs, primer(s) and water (12 µl)

RNA Avg. Ct Std. Dev. %CV 100 ng 24.258 0.072 0.03 100 pg 33.574 0.265 0.79

Add: Buffer

Use up to 50% of the first-strand product directly for qPCR

Input

10

Delta Rn

qScript cDNA SuperMix Protocol 40 minutes

1

0.1 0

5

10

15

20

30

35

40

Fig.2 Reproducibility Cycle Number 48 independent first-strand reactions using 100 ng or 100 pg of HeLa cell total RNA and qScript cDNA SuperMix. 1/10th of each first-strand reaction was used as template for Taq Man qRT-PCR of CDKN1B using PerfeCTa qPCR SuperMix

Add: DTT

Mix and Incubate 2 min at 37oC

14000

3000

Incubate: 10 min at 25oC 50 min at 42oC 15 min at 75oC

10000 8000

-d(RFU)/dT

Normalized RFU

12000 2000

1000

6000 0 80

4000

85 90 Temperature (oC)

95

qScript cDNA SuperMix SuperScript III First Strand System for RT-PCR

2000 0 0 o

Incubate 20 min at 37 C Use only 1/10th of the first-strand product for PCR amplification Fig.1 First Strand Synthesis Protocol Comparison for qScript vs. Conventional Protocol

5

10

15

20 25 Cycle Number

30

35

40

Fig.3 Higher Yield (more accurate representation) of Low Abundance Gene–PP2A PP2A. First-strand synthesis was carried out using 1 µg HeLa cell total RNA with either qScript cDNA SuperMix or SuperScript III cDNA SuperMix. 5 ng total RNA equivalent (1/200th of each cDNA reaction) was used for SYBR Green qPCR of PP2A gene with PerfeCTa SYBR Green SuperMix.

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Reactions

qScript cDNA Synthesis Kit

95047-025 95047-100 95047-500

25 X 20 ul rxns 100 X 20 ul rxns 500 X 20 ul rxns

qScript cDNA SuperMix

95048-025 95048-100 95048-500 95048-096

25 X 20 ul rxns 100 X 20 ul rxns 500 X 20 ul rxns 5 x 96-well plate





qScript Flex cDNA Kit

95049-025 25 X 20 ul rxns 95049-100 100 X 20 ul rxns

cDNA Synthesis

7

qScript XLT One-Step RT-PCR Kit

Tough RT-PCR for Tough Samples

The qScript XLT One-Step RT-PCR kit is for highly sensitive, robust one-step RT-PCR even in the presence of PCR inhibitors often present in crude samples extracted from environmental specimens, plant tissues, or animal tissues.

FEATURES AND BENEFITS ■

Tough tested to overcome inhibitors

Optional Gel Track Loading Dye included Superior reproducibility ■ Convenient single tube reaction assembly at room temperature ■ ■

The qScript XLT One-Step RT-PCR Kit is a convenient and highly sensitive system for amplification of RNA templates up to 2 kb. cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. This system has been optimized to deliver maximum RT PCR efficiency, sensitivity, and specificity. qScript XLT is an engineered M-MLV reverse transcriptase with reduced RNase H activity and improved activity and stability at higher temperatures. The use of higher temperatures (48 to 55°C) for the cDNA synthesis step of one-step RT-PCR provides higher specificity for primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis. The enzyme is supplied as a mixture with ribonuclease inhibitor protein to protect the integrity of RNA templates in crude lysates or samples where RNase contamination may limit assay sensitivity.

8

qScript XLT One-Step RT-PCR Kit

Competitor Q Quanta Competitor L 12341234 1234 L

2058 bp 1502 bp 1052 bp 694 bp

1-Step RT-PCR of varying length amplicons from 2.2-kb TcR in vitro transcript RNA Each kit was used according to the manufacturer’s recommended procedure in 20-µL reaction volumes containing 200 µM each primer and 1 x 105 copies of an in vitro synthesized run-off transcript for the tetracyclin resistance gene (TcR), produced using T7 RNA polymerase. Following first-strand synthesis and activation of the hot-start Taq polymerase, all reactions were amplified for 30 cycles of 94°C, 15s; 60°C, 20s; 72°C, 2 min followed by a final hold of 5 min at 72°C. 1/5th of each reaction was analyzed on a 0.8% agarose, 0.5X TBE gel conatining 0.25 mg/ mL ethidium bromide.

A key component of the system is the One-Step ToughMix. This master mix is highly resistant to PCR inhibitors and contains an ultra pure, hot-start, highly processive thermostable DNA polymerase that is blended with a proof-reading (3’-exonuclease) polymerase for improved PCR fidelity and fragment lengths. High-avidity monoclonal antibodies provide an extremely stringent automatic hot-start that minimizes the potential for primer-dimer and other non-specific PCR artifacts without compromising polymerase activities. Highly specific amplification is crucial to successful RT-PCR as non-specific product(s) can compete with amplification of the target sequence and impair PCR efficiency. The proprietary reaction buffer has been specifically formulated to maximize activities of both the reverse transcriptase and thermostable DNA polymerase while minimizing the potential for primer-dimer and other non-specific PCR artifacts. GelTrack® Loading Dye is a mixture of blue and yellow electrophoresis-tracking dyes that migrate at approximately 4 kb and 50 bp. This optional component simplifies post PCR analysis, allowing direct loading of RT-PCR product on agarose gels following amplification. The GelTrack Loading Dye solution is not included with the sample kit.

1-Step RT-PCR of varying length fragments from HeLa cell total RNA RT-PCR program: 48c 20min; 94C 3min; 94C 15sec; 60C 15 sec; 68C 2min; 35 cycles ACTB = 2 ng HeLa total RNA, all others 20 ng HeLa total RNA Load 5 uL of 20 uL rxn on 0.8% gel (CFB = Complement Factor B, PP2A = protein phosphatase 2A, ACTB = beta actin, APC = adenomatous polyposis coli)

COMPONENTS Reagent Description

Quantity

qScript XLT One-Step 25X concentrated mixture of qScript XLT reverse transcriptase and recombinant Reverse Transcriptase (25X) ribonuclease inhibitor protein. 1 x 200 µL One-Step ToughMix (2X) 2X concentrated reaction buffer containing dNTPs, magnesium chloride, hot-start DNA polymerase, and stabilizers 2 x 1.25 mL GelTrack Loading Dye (50X) 50X concentrated mixture of RT-PCR compatible, blue and yellow 1 x 0.4 mL electrophoresis-tracking dyes Nuclease-free water 2 x 1.5 mL

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size (20uL Reactions)

qScript XLT One-Step RT-PCR Kit

95143-200

200 x 25 µL reactions

qScript XLT One-Step RT-PCR Kit

9

qScript XLT One-Step ToughMixes

TOUGH TESTED RT-qPCR

qScript XLT One-Step RT-qPCR ToughMix is a ready-to-use, highly sensitive master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates. It is a versatile and robust RT-qPCR reagent that provides maximum sensitivity and PCR efficiency with a variety of fluorogenic probe chemistries, including TaqMan® hydrolysis probes.

FEATURES AND BENEFITS Tough Tested — Overcomes common inhibitors including polysacharides, heme/hemoglobin, humic acid, melanin ■ Flexible — Use fast or standard qPCR cycling conditions ■ Broad dynamic range — Reliable data from your precious samples every time ■ Multiplexing enabled — Supports detection of up to four targets ■

DESCRIPTION qScript XLT One-Step RT-qPCR ToughMix is a ready-to-use, highly sensitive master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan 5’-hydrolysis probes on real-time PCR systems that do not require a passive reference dye. First-strand cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. It is ideal for highly sensitive quantification of RNA viruses or low abundance RNA targets as well as high throughput gene-expression studies. The system has been optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity in reduced reaction volumes and fast cycle times. qScript XLT One-Step RT-qPCR ToughMix contains all required components for RT-qPCR except RNA template and probe. It is compatible with all dual-labeled probe chemistries.

10

qScript XLT One-Step ToughMixes

qScript XLT is an engineered M-MLV reverse transcriptase with reduced RNase H activity and improved activity and stability at higher temperatures. The use of higher temperatures (50 to 55°C) for the first-strand step of one-step RT-qPCR provides higher specificity for primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis. qScript XLT One-Step RT-qPCR ToughMix is highly resistant to PCR inhibitors. A key component of the ToughMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This provides an extremely stringent automatic hot-start that minimizes the potential for primer-dimer and other non-specific PCR artifacts. The light blue color of the AccuVue™ tracer dye simplifies reaction assembly in white, or clear, plates and helps to minimize pipetting or mixing errors. It does not interfere with qPCR performance or affect the stability of the product.

1000

6000 5000

Hemoglobin ng/µL

Avg Ct n =3

0 25 50 100 200 400

17.86 17.68 17.73 17.93 18.10 18.50

4000

RFU

RFU

NTC 100 fg 1 pg 10 pg 100 pg 1 ng 10 ng 100 ng

100

3000 2000 1000 0

0

5

10

15

20

25

30

35

40

45

0

5

10

Broad linear dynamic range, low Limit of Detection

15

20

25

30

35

40

Cycle Number

Cycle Number

Enables performance in the presence of inhibitors

INSTRUMENT COMPATIBILITY Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. The qScript XLT One-Step RT-qPCR Kit does not contain a passive reference dye.

ORDERING INFORMATION PRODUCT

Instrument Compatibility

Quanta Cat. No.

Pack Size (20uL Reactions)

qScript XLT 1-Step RT-qPCR ToughMix

Roche LightCycler® 480, Opticon™, Chromo4™, Corbett Rotor-Gene™, eppendorf Mastercycler ®, Bio-Rad CFX

95132-100 95132-500

100 X 20 ul rxns 500 X 20 ul rxns

qScript XLT 1-Step RT-qPCR ToughMix, ROX

AB 7000, 7300, 7700, 7900, StepOne™

95133-100 95133-500

100 X 20 ul rxns 500 X 20 ul rxns

qScript XLT 1-Step RT-qPCR ToughMix, low ROX

AB 7500, Stratagene MX, AB QuantStudio 12K Flex, AB ViiA7, Fluidigm BioMark

95134-100 95134-500

100 X 20 ul rxns 500 X 20 ul rxns

1-step RT-qPCR kits for detection by SYBR Green I:

PRODUCT

Quanta Cat. No. ROX Reference Dye AB 7000, 7300, 7700, 7900 StepOne™

qScript 1-Step SYBR Green RT-qPCR

95088-050 95089-050 95086-050 95087-050 95088-200 95089-200 95086-200 95087-200

Quanta Cat. No. Quanta Cat. No. Low-ROX Reference Dye Fluorecein Reference Dye AB 7500, Stratagene MX Bio-Rad iQ™, MyiQ™, iQ™5 AB QuantStudio 12K Flex, AB ViiA7, Fluidigm BioMark





Quanta Cat. No. Qty. Reactions No Passive Reference Dye (µl/rxn) ® ™ ™ Roche LightCycler 480, Opticon , Chromo4 , ™ ® Corbett Rotor-Gene , Eppendorf Mastercycler , Bio-Rad CFX

50 (50ul) 200 (50ul)

qScript XLT One-Step ToughMixes

11

AccuStart II PCR

SUPERIOR PCR PERFORMANCE

AccuStart II Taq DNA Polymerase and PCR SuperMixes provide high specificity amplification for all PCR applications. The antibody mediated hot-start reaction enables specific and efficient primer extension in the PCR process with the added convenience of room temperature reaction assembly. FEATURES AND BENEFITS Lanes: 1 = XPC 2 = XPC + p53 3 = p53

AmpliTaq Platinum PCR Gold PCR SuperMix Master Mix

1 Mix 2

3

1

2

3

AccuStart II GelTrack PCR SuperMix

1

2

(

3

AmpliTaq Gold 360 Master (

2

(

1

(

High yield, high sensitivity ■ Precise amplification — hot-start technology ensures specific and efficient primer extension ■ GelTrack Loading Dye pre-mixed (GelTrack SuperMix only) ■ Superior reproducibility and precision ■ Convenient reaction assembly at room temperature ■

3

p53 XPC

AccuStart II PCR SuperMix and GelTrack PCR SuperMix AccuStart II PCR SuperMix is a 2X concentrated, ready-to-use reaction cocktail for routine PCR amplification of DNA fragments up to 4 kb. It contains all components, except primers and template. AccuStart II PCR SuperMix simplifies reaction assembly, improves assay reproducibility, and reduces the risk of contamination. A key component is AccuStart II Taq DNA polymerase that contains monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step. Upon heat activation (1 minute at 94°C), the antibodies denature irreversibly, releasing fully active, unmodified Taq DNA polymerase. This enables specific and efficient primer extension with the convenience of room temperature reaction assembly.

12

AccuStart II PCR

Duplicate mouse tail extracts

Superior multiplex PCR genotyping with AccuStart II GelTrack PCR SuperMix

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size

AccuStart II 250U

95141-250

250 X 25 ul rxns

AccuStart II 1000U

95141-01K

1000 X 25 ul rxns

AccuStart II 5000U

95141-05K

5000 X 25 ul rxns

AccuStart II PCR SuperMix

95137-500

500 X 25 ul rxns

AccuStart II PCR SuperMix

95137-04K

4000 X 25 ul rxns

AccuStart II GelTrack PCR SuperMix

95136-500

500 X 25 ul rxns

AccuStart II GelTrack PCR SuperMix

95136-04K

4000 X 25 ul rxns

AccuStart II Mouse Genotyping GENOTYPING WITH HIGH YIELD AND HIGH SPECIFICITY

The AccuStart II Mouse Genotyping Kit is designed for fast and easy preparation of PCR-ready DNA extracts and endpoint PCR analysis from tissues such as tail snips and ear punches commonly used for genotyping knockout and transgenic animals. The kit combines Extracta DNA Prep for PCR - Tissue with AccuStart II GelTrack™ PCR SuperMix in a single kit.

FEATURES AND BENEFITS ■

Simple, fast protocol

■

GelTrack Loading Dye pre-mixed

■

K Mouse Genotyping Kit

Extracta PCR Genotyping Kit

Two mouse tail snips (2.5 mm) were extracted according to the recommended conditions for each kit. The volume of each extract was brought to 300ul and diluted 1/20 with TE buffer. 5ul of diluted extract was used in a 25ul PCR reactions.

Precise amplification — hot-start technology ensures specific and efficient primer extension

■

High yield, high sensitivity

■

Superior reproducibility and precision

AccuStart II Mouse Genotyping Kit Extracta reagents allow rapid extraction of PCR ready DNA that can be used directly in PCR reactions eliminating time consuming or expensive purification steps. AccuStart II GelTrack PCR SuperMix has been optimized for genotyping applications that commonly utilize 3 or more primers in multiplex PCR reactions that allow amplification and analysis of two or more targets in a single reaction such as normal and mutant alleles of a gene knockout or that determine the presence or absence of transgene constructs in transgenic animals.

Contents ■ Extracta DNA Prep for PCR - Tissue, 2 x 25 mL Extraction Reagent and 2 x 25 mL Stabilization Buffer ■ ■

AccuStart II GelTrack PCR SuperMix (2X) 5 x 1.25 mL of 2X reaction mixture containing optimized concentrations of MgCl2, dNTPs, AccuStart II Taq DNA Polymerase, AccuStart Taq antibodies, reaction buffer, stabilizers and gel loading dyes. To reorder individual components, please use the following catalog numbers: 95136-100 95136-500 95136-04K

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size

AccuStart II Mouse Genotyping

95135-500

500 x 25 ul rxns



AccuStart II Mouse Genotyping

13

AccuStart II PCR ToughMix

Tough PCR for Tough Samples

AccuStart II PCR ToughMix is a ready-to-use reaction cocktail for PCR amplification of DNA templates that overcomes many known inhibitors of PCR often present in crude samples extracted from environmental specimens, plant tissues, or animal tissues.

FEATURES AND BENEFITS Robust mix designed to easily overcome PCR inhibitors ■ Stringent antibody hot start ■ Optional Gel Track Loading Dye included ■ Convenient reaction assembly at room temperature

L C

Hemoglobin

Humic Acid

Hematin

Melanin

L

■

AccuStart II PCR ToughMix AccuStart II PCR ToughMix is a 2X concentrated ready-to-use reaction cocktail for the PCR amplification of DNA templates in the presence of inhibitors. It contains all components, except primers and template. A key component of AccuStart PCR ToughMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. These antibodies bind the polymerase and keep it inactive prior to the initial PCR denaturation step. This enables specific and efficient primer extension with the convenience of room temperature reaction assembly. Similar to Taq DNA polymerase, the activated polymerase in AccuStart II PCR ToughMix possesses 5’→3’ DNA polymerase activity and a double-strand specific 5’→3’ exonuclease. The polymerase does not have 3’-exonuclease activity and is free of any contaminating endo or exonuclease activities. PCR products generally contain non-templated dA additions and can be cloned using vectors that have a single 3’-overhanging thymine residue on each end. GelTrack Loading Dye is a mixture of blue and yellow electrophoresistracking dyes that migrate at approximately 4kb and 50 bp. This optional component simplifies post PCR analysis, allowing direct loading of PCR product on agarose gels following amplification. The GelTrack Loading Dye solution is not included with the sample kit.

14

AccuStart II PCR ToughMix

30 cycle PCR; 1 x 10^4 copies TcR DNA (1052-bp amplicon) Inhibitor Resistance of AccuStart II PCR ToughMix A 1-kb fragment from 1e4 copies of the Tetracyclin resistance gene was amplified in 20-µL reaction volumes according to the recommended protocol. Reactions were challenged with varying concentrations of different PCR inibitors as summarized below. Following a 3 min activation at 94°C; PCR was for 30 cycles of: 94°C, 15s; 60°C, 20s; 72°C, 1 min. 1/5th of each reaction was analyzed on a 01% agarose, 0.5X TBE gel conatining 0.25 mg/mL ethidium bromide. Hemoglobin: 316 ng/µL, 100 ng/µL, 31.6 ng/µL, 10 ng/µL, 3.16 ng/µL Humic Acid: 31.6 ng/µL, 10 ng/µL, 3.16 ng/µL, 1 ng/µL, 0.316 ng/µL Hematin: 100 µM, 31.6 µM, 10 µM, 3.16 µM, 1 µM Melanin: 10 ng/µL, 3.16 ng/µL, 1 ng/µL, 0.316 ng/µL, 0.1 ng/µL C: control reactions without inhibitor L: 1 Kb Plus DNA Ladder (Invitrogen)

Dandelion Leaf Extract L C 1 2 3 4 5

Inhibitor Resistance of AccuStart II PCR ToughMix: PCR in the presence of polyphenol spike Varying amounts of a polyphenol-rich plant extract (0.2, 0.06, 0.02, 0.006, or 0.002 uL) were added to 25-uL PCRs containing 10,000 copies of a control template. Amplification was carried out for 30 cycles of: 94°C, 15s; 60°C, 20s; 72°C, 1 min. 1/5th of each reaction was analyzed on a 01%agarose, 0.5X TBE gel containing 0.25 mg/ mL ethidium bromide. As little as 0.002 uL of the crude plant lysate inhibited control reactions with a conventional PCR master mix (data not shown).

30 cycle PCR; 1 x 10^4 copies TcR DNA (1052-bp amplicon)

COMPONENTS AccuStart II GelTrack PCR ToughMix (2X)

2X mix containing optimized concentrations of MgCl2, dNTPs, reaction buffer, hot-start DNA polymerase, stabilizers and gel loading dyes.

GelTrack Loading Dye (50X)

50X concentrated mixture of RT-PCR compatible, blue and yellow electrophoresis-tracking dyes.

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size (20uL Reactions)

AccuStart II PCR ToughMix

95142-800 95142-04K

800 x 25 µL reactions (8 x 1.25 mL) 4000 x 25 µL reactions (1 x 50 mL)



AccuStart II PCR ToughMix

15

AccuStart Genotyping ToughMix DIFFICULT SAMPLES CAUSING UNRELIABLE GENOTYPING RESULTS? WHIP THEM INTO SHAPE WITH THE TOUGHEST PCR MIX AROUND

AccuStart Genotyping ToughMixes enable genetic analysis including TaqMan genotyping directly from crude samples such as lysates, blood spots, plant tissues and clinical samples.

FEATURES AND BENEFITS

COMPARISON TO CONVENTIONAL MASTER MIXES

Bullet-Proof Genotyping — easily overcome tough PCR inhibitors ■ Short Run Times — fast cycling protocols enable more experiments ■ Signal Optimization — robust genotyping with low primer & probe concentration

AccuStart Genotyping ToughMix stands up to the challenge where other genotyping master mixes fall apart. ToughMix can be used with clean templates where it generates higher fluorescent signal and tighter clusters than the leading competitor’s mix (Figure 1A). In the presence of a common the competitor’s system is completely Fig PCR 1A:inhibitor Fast TaqMan Genotyping: BRCA1shut down while ToughMix delivers robust, accurate results (Fig 1B).

■

Flexible Workflow— assembled reactions stable 3 days pre/post-PCR ■

Figure 1A

BRCA1, rs11659028 TAGACAGGGAGCTGGCATGGGCCCT[A/T]GGGAAGTCAAGCTGTAGGTGAGGAT 96-well plate

AccuStart Genotyping ToughMix is a ready-to-use 2X concentrated hot-start PCR mix containing additives which prevent inhibition of PCR or quenching of fluorescent signal by common PCR inhibitors. AccuStart DNA Polymerase is a low DNA hot-start preparation in which residual DNA is undetectable. OVERCOME PCR TROUBLEMAKERS Many sample types are rich in PCR inhibitors (Table 1). These scoundrels operate by shutting down PCR amplification at threshold concentrations, or by quenching fluorescent signal. Expensive or time-consuming purification steps are no longer required to eliminate inhibitors — AccuStart Genotyping ToughMix neutralizes Table 1 problem causing inhibitors in crude lysates and other difficult samples. Table 1

Inhibitor

Polyphenols

Humic Acids Hematin Hemoglobin

Polysaccharides Melanin

16

Common Sources

95C, 5 min 45 cycles: 95C, 5s 60C, 31s 25oC post read

Fig 1B: Reliable Genotyping in Presence of PCR Inhibitors AccuStart Genotyping ToughMix TaqMan GTXpress™ Master Mix

Figure 1B

BRCA1, rs11659028 TAGACAGGGAGCTGGCATGGGCCCT[A/T]GGGAAGTCAAGCTGTAGGTGAGGAT 96-well plate 10-uL Rxn. Vol. 0.5X C___339916_10 primer/probe mix AB 7500 HRC2 gDNA panel (5 ng / rxn) 50 ng/uL humic acid 95C, 5 min 45 cycles: 95C, 5s 60C, 31s

Reagent Performance Competitor

Plant Extracts

AccuStart ToughMix

25oC post read

✔

Soil Plant Tissue

✔

Dried Blood Blood Spots Blood

10-uL Rxn. Vol. 0.5X C___339916_10 primer/probe mix AB 7500 HRC2 gDNA panel (5 ng / rxn)

✔ ✔

Feces Plant Tissue Hair Skin

AccuStart Genotyping ToughMix

✔ ✔ ✔

AccuStart Genotyping ToughMix TaqMan GTXpress Master Mix

LEGENDARY PRODUCT STABILITY

Variability in PCR experiments is often caused by degradation of reagent performance with time and handling. Quanta PCR reagents provide industry-leading stability and will perform for you every time (Fig 2). ■

3 days pre-PCR stability (assembled reactions)

■

5 days post-PCR stability (cycled reactions)

■

2 years shelf-life -20°C

■ ■

Fig 2: BRCA1 Genotyping: AccuStart Genotyping ToughMix Stability > 6 months shelf-life 8°C > 20 freeze-thaw cycles

Figure 2

t=0

3-day Pre-PCR Stability

5-day Post-PCR Stability 6

5

5

5

4

4

4

3

3

3

2

FAM (Y Allele)

6

FAM (Y Allele)

FAM (Y Allele)

6

2

1

1

1 0.0

0.5

1.0

1.5

2.0

2.5

3.0

2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

VIC (X Allele)

VIC (X Allele)

VIC (X Allele)

ORDERING INFORMATION PRODUCT

Instrument Compatibility

Quanta Cat. No.

Pack Size (20uL Reactions)

AccuStart Genotyping ToughMix

Roche LC480, eppendorf (all) Illumina ECO, Bio-Rad (all) Qiagen/Corbett Rotorgene (all)

95115-250 95115-012 95115-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

AccuStart ABI 7000, 7300, 7700, 7900 (all) Genotyping StepOne (all) ToughMix ROX

95116-250 95116-012 95116-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

AccuStart Genotyping ToughMix Low ROX

ABI ViiA7, 7500 Stratagene MX (all) AB QuantStudio 12K Flex, Fluidigm BioMark

95117-250 95117-012 95117-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

Related Products Extracta DNA Prep for PCR

Direct lysis solution for DNA Preparation from mammalian tissues

95091-025 95091-250

25 mL 250 mL



AccuStart Genotyping ToughMix

17

AccuMelt HRM — High Resolution Melt Analysis SuperMix ACCURATELY CHARACTERIZE GENETIC VARIATIONS FROM NOVEL MUTATIONS TO RARE POLYMORPHISMS AND MORE

AccuMelt HRM SuperMix maximizes differences in melt temperature and curve shape to allow discrimination of DNA sequence differences amongst different samples. AccuMelt is suitable for a broad range of applications including SNP analysis, mutation scanning, transgene analysis, species identification and DNA methylation analysis.

FEATURES AND BENEFITS

Superior Resolution of Genotypes

See sequence differences clearly — robust amplification ensures sufficient yield of products to generate discrete melt curves

SNP Genotyping is a useful application for HRM and illustrates the capabilities of AccuMelt HRM SuperMix. Genotypes are readily identified based on unique melting profiles depending on a sample’s sequence (Figure 1). Furthermore, AccuMelt HRM SuperMix gives superior resolution of difficult genotypes when compared to the leading competitor’s mix based on greater Tm differences observed for A → T transversions (Figure 2).

■ Accurate genotype calling — comparable or better performance than TaqMan Genotyping

Work with rare or precious samples — large range of template inputs possible ■

■ Specificity — works with lower Mg2+ concentration than other systems thus enhancing assay accuracy

AccuMelt HRM SuperMix AccuMelt HRM SuperMix is a ready-to-use 2X concentrated hotstart PCR mix containing SYTO 9™ green fluorescent DNA-binding dye. AccuStart Taq DNA Polymerase allows for room-temperature reaction assembly and storage at +4°C for 6 months.

Panel A

100

T, Tm = 82.90 A, Tm = 83.21 C, Tm = 83.56 G, Tm = 83.96

80

Normalized (-dRFU/dT)

■

60 40 20 0 79

80

81

82

83

84

85

86

87

Temperature (oC) Panel B

Fig.1 High resolution melting analysis of a model SNP system with a single A,C,G,or T variant base. AccuMelt HRM SuperMix readily resolves each genotype and Tm differences are easily visualized in normalized melting curve plots (Roche, Lightcycler 480).

Normalized (-dRFU/dT)

100

T, Tm = 81.64 A, Tm = 81.85 C, Tm = 82.26 G, Tm = 82.57

80 60 40 20 0 77

78

79

80

81

82

83

84

85

86

Temperature (oC) Fig.2 Effect of T,A,C, or G variant base on Tm in a model HRM SNP system with either AccuMelt HRM SuperMix (Panel A), or a competitor’s SYTO 9 dye master mix (Panel B). Plots of averaged melt peaks normalized to maximum signal for each system.

18

AccuMelt HRM — High Resolution Melt Analysis SuperMix

COMPARISON TO TAQMAN GENOTYPING

B

Fig.3 Accuracy of HRM genotyping with AccuMelt HRM SuperMix was evaluated by comparison to TaqMan detection of the G>A rs1801133 SNP in the MTHFR gene. Panel A) HRM normalized melting curves; Panel B) TaqMan allelic discrimination plots. TaqMan failed to resolve Sample D3 (labeled as “UNKN”) which was typed as a heterozygote by HRM.

COMPARISON TO TAQMAN GENOTYPING

ROBUST AMPLIFICATION

TaqMan Genotyping has been used successfully in SNP analysis and other allelic discrimination applications. This widely adopted standard in genotyping was used as a benchmark to assess the utility of HRM with our SuperMix. AccuMelt HRM was determined to be just as effective as TaqMan Genotyping in SNP analysis and was even able to call the genotype for a difficult sample which the TaqMan assay could not resolve (see Figure 3).

Consistent robust amplification is critical to accuracy in HRM analysis. AccuMelt HRM SuperMix will drive all PCR amplifications to plateau regardless of the quantity of template input (Figure 4). This ensures accurate results regardless of the quantity of DNA available.

Fluorescence (483 - 533)

ORDERING INFORMATION PRODUCT Quanta Cat. No. Pack Size AccuMelt HRM SuperMix



95103-250 95103-012 95103-05K

120

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

AccuMelt HRM SuperMix Leading SYBR Green Master Mix

100 80

slope r int. eff. (%)

60

-3.343 -0.9996 31.91 99.11

-3.340 -0.994 30.71 99.27

40 20 0 0

5

10

15

20

25

30

35

40

45

Cycles Quanta Biosciences, AccuStart and AccuMelt are trademarks of Quanta Biosciences. SYTO 9 ® and SYBR are registered trademarks of Molecular Probes, Inc. TaqMan® and LightCycler ® are registered trademarks of Roche Molecular Systems, Inc. Quanta BioSciences is licensed for PCR.

Fig.4 High yield, high efficiency PCR with AccuMelt HRM SuperMix. Real-time PCR of GAPDH was amplified from log-fold serial dilutions of qScript synthesized cDNA from HeLa cell total RNA (10 ng to 0.1 pg) was carried out with either a leading SYBR Green Master Mix or AccuMelt HRM SuperMix using the following cycling conditions: 95°C, 20s; followed by 45 cycles of: 95°C, 3s; 60°C, 20s. Averaged plots for quadruplicate reactions for each input quantity are shown.

AccuMelt HRM — High Resolution Melt Analysis SuperMix

19

PerfeCta PreAmp SuperMix

UNBIASED PRE-AMPLIFICATION

High order multiplexed enrichment of specific sequences from limiting template using low-cycle PCR

FEATURES AND BENEFITS ■ ■ ■

5X concentrated SuperMix for lower reaction volumes / higher sample volumes Adding more value to your precious samples AccuStart II hot-start

Extract / Purify RNA

1st strand cDNA synthesis

Assemble PreAmp Reaction

n

PreAmp SuperMix (5X) Pooled primers / probe assay sets (~50 nM ea primer) cDNA (from 100 pg to 250 ng total RNA) Water q.s. 50 µL

AAA

AAA nAAAAAA

AAAAAAn An AA

A AA AAAAAAn

qScript cDNA SuperMix

PreAmp Process Flow: 1. Prepare RNA 2. Reverse transcribe RNA

10 to 14 cycles preamplification * Optional * Exo I removal of PreAmp primers

3. Pool assay primers and dilute 4. Perform pre-amplification reaction

Dilute PreAmp product

5. Dilute PreAmp reaction product Assemble individual real-time qPCRs: probe assays or SYBR Green

PerfeCTa® qPCR ToughMix® PerfeCTa® qPCR FastMix® II

PerfeCTa® SYBR® Green FastMix® PerfeCTa® SYBR® Green SuperMix

Real-time qPCR (40 - 45 cycles)

Analyze Results

20

PerfeCta PreAmp SuperMix

6. Perform individual qPCRs for each pre-amplified gene of interest (GOI).

Fig.2A 96-gene Cq comparison for control versus 10-cycle pre-amplified cDNA. cDNA prepared from Universal Reference RNA, either 100 ng, 10 ng, or 1 ng (total RNA equivalent), were pre-amplified for 10 cycles in 20-uL reaction volumes. Following pre-amplification, product was diluted 5-fold and 0.5 uL used as template in individual qPCRs for each gene using PerfeCta SYBR Green SuperMix. Assuming 1,000-fold enrichment: 500 ng, 50 ng, or 5 ng of PreAmp product was used as template in each 10-uL qPCR. Cq for each amount of pre-amplified cDNA (Y-axis) are plotted against Cq obtained for 10 ng of control (no PreAmp) cDNA (X-axis). Idealized plots representing perfect (100%) pre-amplification efficiency for each input amount are shown in black.

Fig.2B 96-gene Cq comparison for control versus 14-cycle pre-amplified cDNA. cDNA prepared from Universal Reference RNA, either 10 ng, 1 ng, or 0.1 ng (total RNA equivalent), were pre-amplified for 14 cycles in a 20-uL reaction volume. Following pre-amplification, product was diluted 10-fold and 0.5 uL used as template in individual qPCRs for each gene using PerfeCta SYBR Green SuperMix. Assuming 16,000-fold enrichment: 400 ng, 40 ng, or 4 ng of PreAmp product, was used as template in each 10-uL qPCR. Cq for each amount of pre-amplified cDNA (Y-axis) are plotted against Cq obtained for 10 ng of control (no PreAmp) cDNA (X-axis). Idealized plots representing perfect (100%) pre-amplification efficiency for each input amount are shown in black.

PerfeCta PreAmp SuperMix is a 5X concentrated, ready-to-use reaction cocktail for unbiased, selected enrichment of target sequences from limiting amounts of starting material for downstream gene expression profiling or targeted re-sequencing. It contains all components, except primers and templates. The 5X concentrated SuperMix allows addition of higher template volumes when working with low concentration samples, and/or reduced reaction volumes. Inclusion of an inert light blue tracer dye helps visualize small reaction volumes and ensure accurate pipetting. PerfeCta PreAmp SuperMix delivers unbiased pre-amplification of up to 100 target sequences from as little as 100 pg of total cDNA. It is compatible with both TaqMan 5’-nuclease probes or ds-DNA binding dye (i.e. SYBR Green I) qPCR detection chemistries. A key component of PerfeCta PreAmp SuperMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This proprietary polymerase mix is resistant to PCR inhibitors and provides an extremely stringent automatic hot-start allowing reaction assembly, and temporary storage, at room temperature prior to pre-amplification.

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size (20uL Reactions)

PerfeCta PreAmp SuperMix

95146-040

40 x 50-μL reactions

PerfeCta PreAmp SuperMix

21

qPCR SuperMixes and FastMixes– SYBR Green PERFECTA QPCR SUPERMIXES AND FASTMIXES FOR EFFICIENT, SENSITIVE AND PRECISE QPCR USING SYBR GREEN DETECTION

PerfeCTa SYBR Green SuperMixes set a new standard for fast and reproducible quantitative PCR (qPCR) with proprietary buffers and SYBR Green stabilizers that maximize fluorescent signal, PCR efficiency, and reduce primer dimers. Rigorous functional testing ensures a broad dynamic range and high sensitivity on a variety of templates, giving unparalleled performance that you can rely on.

FEATURES AND BENEFITS FastMixes–Offer shorter run times enabling more experiments per day ■

Broad Dynamic Range–More reliably attain useful data from your precious samples

■

Superior Antibody-Mediated Hot-Start–Higher specificity leading to more accurate quantification ■

Fast Cycling–Use existing primer sets, no need to re-optimize ■

PerfeCTa SYBR Green SuperMixes PerfeCTa SYBR Green SuperMixes are 2X concentrated, ready-to-use reaction cocktails containing all components, except primers and template for quantitative PCR. Proprietary buffers and stabilizers are optimized for SYBR Green I dye to deliver maximum efficiency, sensitivity, and robust fluorescent signal compared with next-generation competitor kits (See Figures 1 & 2). PerfeCTa SYBR Green SuperMixes are available and optimized for all real-time PCR instrument platforms including those requiring normalization with ROX reference dye or fluorescein (Bio-Rad). These SuperMixes provide the highest level of specificity to reduce the occurrence or delay the detection of primer-dimer and other non-specific artifacts. A key component of these SuperMixes is our AccuStart Taq DNA polymerase, which enables specific and efficient primer extension with the convenience of room temperature reaction assembly. AccuStart Taq DNA polymerase contains monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step. Upon heat activation the antibodies denature irreversibly, releasing fully active and unmodified Taq DNA polymerase.

22

qPCR SuperMixes and FastMixes— SYBR Green

PerfeCTa SYBR Green FastMixes PerfeCTa SYBR Green FastMixes set a new standard for high efficiency qPCR results with a significantly faster protocol for increased productivity using your existing primer sets. PerfeCTa SYBR Green FastMixes are 2X concentrated, readyto-use reaction mixes delivering maximum PCR efficiency, sensitivity, specificity and robust fluorescent signal using either fast or conventional cycling protocols (See Figures 3 & 4). Rapid cycling is achieved by instant activation of AccuFast™ Taq DNA polymerase coupled with rapid polymerization kinetics. Achieve high performance qPCR in as little as 33 minutes. PerfeCTa SYBR Green FastMixes are available and optimized for all real-time PCR instrument platforms including those requiring normalization with ROX reference dye or fluorescein (Bio-Rad). Improved Productivity with PerfeCTa SYBR Green FastMix

Conventional Reagents Cycling

PerfeC Ta FastMix Cycling

Activation:

2-15 min, 95°C

20s, 95°C

PCR Cycling:

15s, 95°C 60s, 60°C

1-3s, 95°C 20s, 60°C

Cycle Time:

40 cycles=52 min (excluding ramp time)

40 cycles=14 min (excluding ramp time)

Total Run Time: iCycler iQ

1 hr, 20 min – 1 hr, 43 min (4-6 runs/8 hr shift)

40 min (12 runs/8 hr shift)

Total Run Time: eppendorf Mastercycler, ep realplex

1 hr, 12 min – 1 hr, 22 min (5-6 runs/8 hr shift)

33 min (14 runs/8 hr shift)

PERFECTA SYBR GREEN SUPERMIXES AND FASTMIXES: PERFORMANCE DATA 35

25 20 15

Slope -3.252 -3.400 R2 -0.9973 -0.9933 PCR Eff. 103% 96.9% 0.001

0.01 0.1 1.0 10 Initial Quantity (ng total RNA)

100

0

5

10

15

1

20

25

Cycle Number

30

35

40

5

10

20

25

30

35

40

45

Fig.2 Superior Reproducibility and Sensitivity PKCA target amplified from log-fold serial dilutions of qScript synthesized cDNA from HeLa cell total RNA (100 ng-1 pg) with either PerFeCTa SYBR Green SuperMix or SYBR GreenER™ qPCR SuperMix. Slope -3.266 -3.400 R2 -0.997 -0.988 PCR Eff. 102.4% 92.2%

34

4000

28 26

3000

24

1 2 3 4 5 Log Initial Quantity (pg HeLa total RNA)

15

NTC

Cycle Number

36

22

2000

PerfeCTa SYBR Green SuperMix SYBR GreenER™ qPCR SuperMix

0

30

20

0.001 0.01 0.1 1.0 10 100 Initial Quantity (ng total RNA)

0.1

RFU

RFU

3000

Cycle Threshold

4000

32

Slope -3.252 -3.554 R2 -0.9973 -0.9970 PCR Eff. 103% 91.1 %

Failed qPCR

Slope -3.266 -3.305 R2 -0.997 -0.996 PCR Eff. 102.4% 100.7%

34

25 20

Fig.1 Superior Reproducibility and Sensitivity PKCA target amplified from log-fold serial dilutions of qScript synthesized cDNA from HeLa cell total RNA (100 ng-1 pg) with either PerfeCTa SYBR Green SuperMix or Power SYBR Green PCR Master Mix 36

30

Failed qPCR 1 pg cDNA

PerfeCTa SYBR Green SuperMix Power SYBR Green PCR Master Mix

0.1

Cycle Threshold

30

Cycle Threshold

1

35

10

Delta Rn

Cycle Threshold

Delta Rn

10

32 30 28 26 24 22 20

2000

1 2 3 4 5 Log Initial Quantity (pg HeLa total RNA)

PerfeCTa SYBR Green FastMix

PerfeCTa SYBR Green FastMix QuantiFast SYBR Green PCR Kit

iQ™ SYBR Green Supermix

1000

1000

0

0 0

5

10

15

20

25

30

35

40

0

5

10

Fig.3 Faster, More Sensitive ADAR target amplified from log-fold serial dilutions of qScript synthesized cDNA from HeLa cell total RNA (100 ng-10 pg) with either PerfeCTa SYBR Green FastMix or QuantiFast SYBR Green PCR Kit.

15

20

25

30

35

40

Cycle Number

Cycle Number

Fig.4 Faster, More Sensitive ADAR target amplified from log-fold serial dilutions of qScript synthesized cDNA from HeLa cell total RNA (100 ng-10 pg) with either PerFeCTa SYBR Green FastMix or iQ SYBR Green Supermix.

O R D E R I N G I N F O R M ATI O N PerfeCTa SYBR Green SuperMixes and PerfeCTa SYBR Green FastMixes are formulated with and without reference dye to provide trouble-free compatibility with specific real-time PCR instruments as indicated below.

PRODUCT

Quanta Cat. No. Quanta Cat. No. Low-ROX Reference Dye Fluorecein Reference Dye AB 7500, Stratagene MX Bio-Rad iQ™, MyiQ™, iQ™5 AB QuantStudio 12K Flex, AB ViiA7, Fluidigm BioMark



SYBR Green qPCR

PerfeCta SYBR Green SuperMix

SYBR Green qPCR w/UNG

PerfeCta SYBR Green SuperMix UNG

Fast Cycling SYBR Green qPCR PerfeCta SYBR Green FastMix



Quanta Cat. No. ROX Reference Dye AB 7000, 7300, 7700, 7900 StepOne™

Quanta Cat. No. Qty. Reactions No Passive Reference Dye (µl/rxn) Roche LightCycler 480, Opticon™, Chromo4™, Corbett Rotor-Gene™, eppendorf Mastercycler, Bio-Rad CFX

95055-100 95056-100 95053-100 95054-100 95055-500 95056-500 95053-500 95054-500 95055-02K 95056-02K 95053-02K 95054-02K

100 (50µl) 500 (50µl) 2000 (50µl)

95069-100 95070-100 95067-100 95068-100 95069-500 95070-500 95067-500 95068-500 95069-02K 95070-02K 95067-02K 95068-02K

100 (50µl) 500 (50µl) 2000 (50µl)

95073-250 95074-250 95071-250 95072-250 95073-012 95074-012 95071-012 95072-012 95073-05K 95074-05K 95071-05K 95072-05K

250 (20µl) 1250 (20µl) 5000 (20µl)

qPCR SuperMixes and FastMixes— SYBR Green

23

PerfeCta FastMix II IMPROVED SENSITIVITY AND REPRODUCIBILITY

PerfeCta FastMix II is an advanced qPCR reagent system for both fast and conventional PCR cycling protocols or instruments. It is a versatile and robust solution that provides the ultimate sensitivity and high PCR efficiency using a variety of fluorogenic probe chemistries, including TaqMan hydrolysis probes.

FEATURES AND BENEFITS ■ ■

■

Shorter run times enabling more experiments per day Broad dynamic range – more reliably attain useful data from your samples Superior antibody-mediated hot-start – higher specificity

DESCRIPTION PerfeCta FastMix II is an advanced qPCR reagent system for both fast and conventional PCR cycling protocols or instruments. It is a versatile and robust solution that provides the ultimate sensitivity and high PCR efficiency using a variety of fluorogenic probe chemistries, including TaqMan hydrolysis probes. PerfeCta FastMix II is provided as a 2X concentrated ready-to-use reaction cocktail that contains all required reaction components, except primers, probe(s), and DNA template. The light blue color of the AccuVue tracer dye simplifies reaction assembly in white, or clear, plates and helps to minimize pipetting or mixing errors. It does not interfere with qPCR performance or affect the stability of the product.

24

PerfeCta FastMix II

A key component of PerfeCta FastMix II is an ultra pure, processive thermostable DNA polymerase that is free of detectable E. coli DNA. PerfeCta FastMix II is ideal for demanding qPCR applications such as bacterial pathogen detection where residual host DNA in typical recombinant enzyme preparations can limit assay sensitivity and obscure detection of low copy samples. The enzyme in PerfeCta FastMix II is combined with high avidity monoclonal antibodies to provide a stringent automatic hot-start that allows reaction assembly, and temporary storage, at room temperature prior to PCR amplification.

Log-fold dilutions: 20ng to 20pg and NTC triplicate qPCRs for each input quantity

ORDERING INFORMATION PRODUCT

Instrument Compatibility

Quanta Cat. No.

Pack Size

PerfeCta FastMix II

Roche LightCycler 480, Opticon, Chromo4, Corbett Rotor-Gene, eppendorf Mastercycler, Bio-Rad CFX

95118-250 95118-012 95118-05K

250 x 20 µl rxns 1250 x 20 µl rxns 5000 x 20 µl rxns

PerfeCta FastMix II ROX

AB 7000, 7300, 7700, 7900, StepOne 95119-250 95119-012 95119-05K

250 x 20 µl rxns 1250 x 20 µl rxns 5000 x 20 µl rxns

PerfeCta FastMix II Low ROX AB 7500, Stratagene MX, AB QuantStudio 95120-250 12K Flex, AB ViiA7, Fluidigm BioMark 95120-012 95120-05K

250 x 20 µl rxns 1250 x 20 µl rxns 5000 x 20 µl rxns



PerfeCta FastMix II

25

PerfeCta qPCR ToughMix DIFFICULT SAMPLES MAKING YOUR PCR UNRELIABLE? WHIP YOUR GENE EXPRESSION EXPERIMENTS INTO SHAPE WITH THE TOUGHEST MIX AROUND

PerfeCta qPCR ToughMixes enable expression analysis and other quantitative applications from crude samples such as lysates, blood spots, plant tissues and clinical samples.

FEATURES AND BENEFITS

COMPARISON TO CONVENTIONAL MASTER MIXES

Bullet-Proof qPCR — easily overcome tough PCR inhibitors Short Run Times — fast cycling protocols enable more experiments ■ Optical Clarity Technology — high precision in reduced reaction volumes ■ Repeatable Results — hot-start polymerase highly stable at ambient temperature

PerfeCta qPCR ToughMix stands up to the challenge where other qPCR master mixes fall apart. qPCR is performed on clean template and in the presence of PCR inhibitors with PerfeCta qPCR ToughMix and three relevant competitor’s master mixes (Figure 1). Average CT values obtained with PerfeCta ToughMix are unaffected by the presence of the PCR inhibitors while competitor’s mixes show signs of inhibition and in some cases fail to amplify.

■ ■

PerfeCta qPCR ToughMix is a ready-to-use 2X concentrated hotstart PCR mix containing additives which prevent inhibition of PCR or quenching of fluorescent signal by common PCR inhibitors. AccuFast DNA Polymerase is a low DNA hot-start preparation in which residual DNA is undetectable. OVERCOME PCR TROUBLEMAKERS Many sample types are rich in PCR inhibitors (Table 1). These PCR scoundrels operate by shutting down PCR amplification at threshold concentrations, or by quenching fluorescent signal. Expensive or timeconsuming purification steps are no longer required to eliminate inhibitors — PerfeCta qPCR ToughMix neutralizes problem-causing inhibitors in crude lysates and other difficult samples.

Table 1 Table 1

Inhibitor

Polyphenols

Humic Acids Hematin Hemoglobin

Polysaccharides Melanin

26

PerfeCta qPCR ToughMix

Common Sources

Reagent Performance Competitor

Plant Extracts

✔

Soil Plant Tissue

✔

Dried Blood Blood Spots Blood

Feces Plant Tissue Hair Skin

PerfeCTa ToughMix

✔ ✔

✔ ✔ ✔

Effect of PCR Inhibitors on qPCR of MYC cDNA

PerfeCTa qPCR ToughMix

Average Cycle Threshold (CT)

40

Path-ID™ qPCR Master Mix

40

40

35

35

35

30

30

30

25

25

25

20

20

20

15

2 pg

20 pg

200 pg

2 ng

15

20 ng

2 pg

Input Quantity

20 pg

200 pg

2 ng

TaqMan Environmental Master Mix 2.0

20 ng

15

2 pg

20 pg

Input Quantity

200 pg

2 ng

20 ng

Input Quantity

Slope

r

Int.

Eff.

Slope

r

Int.

Eff.

Slope

r

Int.

Eff.

-3.365 -3.267 -3.420

-0.9989 -0.9970 -0.9988

27.57 27.42 28.08

98.2% 102.4% 96.1%

-3.376 -3.935 -3.258

-0.9968 -0.9953 -0.9989

29.07 33.93 29.32

97.7% 79.5% 102.7%

-3.409 -3.616 -3.480

-0.9968 -0.9965 -0.9989

28.14 30.77 28.92

96.5% 89.0% 93.8%

ORDERING INFORMATION PRODUCT

Instrument Compatibility

Quanta Cat. No.

Pack Size

PerfeCta qPCR ToughMix

Roche LC480, eppendorf (all), Illumina ECO Qiagen/Corbett Rotorgene (all), Bio-Rad (all)

95112-250 95112-012 95112-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

PerfeCta qPCR ToughMix UNG

95138-250 95138-012 95138-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

PerfeCta qPCR ToughMix ROX

95113-250 95113-012 95113-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

PerfeCta qPCR ToughMix UNG ROX

95139-250 95139-012 95139-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

PerfeCta qPCR ToughMix Low-ROX

95114-250 95114-012 95114-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns

PerfeCta qPCR ToughMix UNG Low-ROX 95140-250 95140-012 95140-05K

250 X 20 ul rxns 1250 X 20 ul rxns 5000 X 20 ul rxns





ABI 7000, 7300, 7700, 7900 (all) StepOne (all)

ABI ViiA7, 7500 Stratagene MX (all) Fluidigm BioMark, AB QuantStudio 12K Flex

PerfeCta qPCR ToughMix

27

PerfeCta MultiPlex qPCR ToughMix

qPCR multiplexing for tough samples

PerfeCta MultiPlex qPCR ToughMix is a multiplexing technology capable of overcoming many known inhibitors of PCR often present in crude samples extracted from

FEATURES AND BENEFITS

RFU

environmental specimens, plant tissues, or animal tissues.

Tough Tested - Overcomes common inhibitors including polysacharides, heme/hemoglobin, humic acid, melanin ■

Flexible - Use fast or standard qPCR cycling conditions Broad dynamic range - Reliable data from your precious samples every time ■ ■

The PerfeCta Multiplex qPCR ToughMix, capable of overcoming many known inhibitors, transcends the multiplex limitations of conventional PCR master mixes, enabling unbiased amplification of up to five target sequences in a single tube. Suppression of low copy amplicons by high copy reference targets in the amplification is a common problem in multiplex PCR that can skew, or mask the apparent representation and quantification of low copy target sequences. PerfeCta MultiPlex qPCR ToughMix results in multiplexed qPCR with dynamic range and sensitivity that are comparable to single-plex qPCR probe assays without the need for limiting or variable primer concentrations. PerfeCta MultiPlex qPCR ToughMix is a 5X concentrated, readyto-use reaction cocktail for real-time quantitative PCR (qPCR). It contains all components, except primers, probes and templates. The 5X concentrated ToughMix allows addition of higher amounts of template and improved detection sensitivity with low concentration samples. PerfeCta MultiPlex qPCR ToughMix has been optimized to deliver maximum PCR efficiency, sensitivity, and specificity in reduced reaction volumes with fast cycle or conventional PCR cycling protocols.

28

PerfeCta MultiPlex qPCR ToughMix

High efficiency, high sensitivity multiplex qPCR results with PerfeC ta MultiPlex qPCR ToughMix Log-fold serial dilutions (10 to 1 E7 copies) of a plasmid containing the GAPDH gene, as well as no template controls, were amplified with PerfeCta MultiPlex qPCR ToughMix as either a singleplex qPCR, or a 4-target multiplexed qPCR that contained 1 E8 copies of 3 additional plasmid DNAs (ACTB, IL1beta, and TUBA). Quadruplicate reactions for each input quantity were carried out in 25-uL volumes with 300 nM each primer and 150 nM each probe. Dual-labeled probes with non-fluorescent quenchers were from Biosearch Technologies. GAPDH was detected using a FAM-BHQ1 probe. ACTB; CAL Fluor Orange 560-BHQ1; IL1beta: CAL Fluor Red 610-BHQ2; TUBA: Quasar 670 – BHQ3. Single-plex qPCRs only contained the GAPDH primers and probe. Cycling was performed on a Bio-Rad CFX with the following protocol. 95C, 2 min; followed by 40 cycles of 95C, 10s; 58C, 90s. RFU data were exported to Excel, averaged for each replicate reaction series, and plotted.

COMPONENTS A key component of PerfeCta MultiPlex qPCR ToughMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This proprietary polymerase mix is highly resistant to PCR inhibitors and provides an extremely stringent automatic hot-start allowing reaction assembly, and temporary storage, at room temperature prior to PCR amplification. PerfeCta Multiplex qPCR ToughMix

5X reaction buffer containing optimized concentrations of MgCl2, dNTPs (dATP, dCTP, dGTP, dTTP), hot-start DNA polymerase, and stabilizers.

PerfeCta Multiplex qPCR ToughMix ROX

5X reaction buffer containing optimized concentrations of MgCl2, dNTPs (dATP, dCTP, dGTP, dTTP), hot-start DNA polymerase, ROX reference dye and stabilizers.

PerfeCta Multiplex qPCR ToughMix Low ROX 5X reaction buffer containing optimized concentrations of MgCl2, dNTPs (dATP, dCTP, dGTP, dTTP), hot-start DNA polymerase, ROX reference dye and stabilizers. INSTRUMENT COMPATIBILITY Different real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is important to match the appropriate reference dye to each specific optical detection system. PerfeCta MultiPlex qPCR ToughMix does not contain an internal reference dye to allow greater flexibility in your choice of reporter fluorophores and instrument platforms. Your choice of probe reporter dyes and any optional internal reference dye must be matched to the excitation and emission optics of your particular instrument. PerfeCta qPCR ToughMix, ROX contains an optimal concentration of a stabilized carboxy-X-rhodamine compound (ROX™) for instruments that use an excitation wavelength of ~490 nm and 605 to 610 nm emission channel for the reference signal. PerfeCta MultiPlex qPCR ToughMix Low ROX contains a ROX reference dye designed for qPCR systems using a 580 nm to 585 nm excitation wavelength for the ROX dye channel, such as Applied Biosystems 7500, 7500 Fast, ViA™ 7, or Stratagene MX4000™, MX3005P™, MX3000P™. It can also be used on real-time PCR systems that do not require a passive reference. Please visit our Product Finder selection tool at www.quantabio.com to find the correct product for your real-time PCR system. STORAGE AND STABILITY PerfeCta Multiplex qPCR ToughMix is stable for 2 years when stored in a constant temperature freezer a. 20ºC, protected from light. For convenience, it may be stored unfrozen at +2 to +8ºC for up to 6 months. Repeated freezing and thawing of the ToughMix will not affect product performance.

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size (20uL Reactions)

Perfecta Multiplex qPCR ToughMix

95147-250 95147-01K 95147-05K

250 x 25 ul rxns 1000 x 25 ul rxns 5000 x 25 ul rxns

Perfecta Multiplex qPCR ToughMix, ROX

95148-250 95148-01K 95148-05K

250 x 25 ul rxns 1000 x 25 ul rxns 5000 x 25 ul rxns

Perfecta Multiplex qPCR ToughMix, Low ROX

95149-250 95149-01K 95149-05K

250 x 25 ul rxns 1000 x 25 ul rxns 5000 x 25 ul rxns







PerfeCta MultiPlex qPCR ToughMix

29

qScript microRNA Quantification System DETECT AND QUANTIFY microRNAs WITH SUPERIOR SENSITIVITY

Quanta’s miRNA profiling system includes the most sensitive and accurate kits and assays for RT-qPCR of microRNA signatures.

FEATURES AND BENEFITS

CONFIDENCE IN YOUR RESULTS

Detect microRNAs with log-fold greater sensitivity than currently available systems ■ Profile many microRNAs from the same cDNA sample ■ Integrated system includes positive control and does not require expensive hydrolysis probes

Quanta’s technology allows inclusion of a minus-poly(A) polymerase control not available with other protocols. The minus-PAP control provides confidence in detection of low-abundance miRNAs, giving you reliable results. Each kit also includes a positive control for SNORD44 that may also be used as a calibration target.

■

■

Broad dynamic range of RNA input

Profile your targets – hundreds of microRNA assays available and growing

Figure 1

■

Poly(A) Tailing

5’

Poly(A) Polymerase

MicroRNA

AAAAAAAAAAAAAAA

QUANTA microRNA Technology The qScript microRNA cDNA Synthesis Kit may be used with total RNA or preparations pre-enriched for miRNAs. First a poly(A) tail is added to miRNAs followed by cDNA synthesis using an adapter primer and qScript RT in a single-tube reaction. The resulting cDNA is ready for qPCR amplification with our Universal PCR Primer and your choice of miRNA assays. This unique single-tube system provides ease-of-use and accurate quantification of microRNAs with a high degree of sensitivity and specificity (Figure 1). PerfeCta SYBR Green SuperMixes ensure compatibility with any real-time thermal cycler.

DETECTION OF RARE microRNAs QUANTA’s microRNA profiling system provides linear detection and quantification of miRNAs across total-RNA input levels spanning six orders of magnitude (Figure 2). This means miRNAs will be detected even when tissue is scarce or the miRNAs are rare. The Quanta miRNA system will detect low copy miRNAs more reliably than other systems and due to the minus-PAP control you will have confidence in the results.

30

qScript microRNA Quantification System

ATP

3’

AAAAAAAAAAAAAAA

cDNA Synthesis

Oligo-dT Adapter Primer

dNTPs Reverse Transcriptase

AAAAAAAAAAAAAAA

microRNA Assay Primer qRT-PCR with SYBR Green SuperMix Universal PCR Primer

Figure 2

Figure 3 miR-124a

SUPERIOR SENSITIVITY QUANTA’s miRNA Profiling System yields superior results when compared to other leading miRNA quantification systems (Figure 3). The qScript microRNA cDNA Synthesis Kit, PerfeCta microRNA-specific assays and PerfeCta SYBR Green SuperMix form an integrated system that yields industry-leading results. PerfeCta microRNA-SPECIFIC ASSAYS & CONTROLS

Cycles

QUANTA’s PerfeCta microRNA assays are lab-validated SYBR green assays and are specific to

mir-27a

the miRNA sequences in miRbase. Each consists of a single miRNA-specific primer (Figure 1) that works in conjunction with the Universal PCR Primer to form a robust and specific assay. Each qScript microRNA cDNA Sytnthesis Kit contains both the Universal PCR Primer and Positive Control Primer, which are also available individually (Table 1). ACCURATE PROFILING IN ANY TISSUE TYPE The qScript microRNA Quantification System accurately detects present miRNAs in any tissue type with correlation to established values (Figure 4). Relative abundance of miRNAs is established using any of several small RNA controls as reference points. This makes miRNA profiling amongst different samples and tissue types straightforward.

Cycles

Let 7a

Figure 4 Relative Abundance of MicroRNAs in Human Tissues Cycles

Relative Abundance of MicroRNAs in Human Tissues

ORDERING INFORMATION PRODUCT

Quanta Cat. No.

Pack Size

PRODUCT

Quanta Cat. No.

Pack Size

qScript microRNA cDNA Synthesis Kit

95107-025

25 rxn

100 x 50uL rxn

100 rxn

PerfeCta SYBR Green SuperMix ROX

95055-100

95107-100

95055-500

500 x 50uL rxn

(for all AB platforms

95055-02K

2000 x 50uL rxn

PerfeCta microRNA- Specific Assay

Contact your VWR Life Science Specialist

500 assays

except 7500) 100 x 50uL rxn

95109-250

250 x 50uL assays

PerfeCta SYBR Green SuperMix Low-ROX

95056-100

PerfeCta Universal microRNA Primer

95056-500

500 x 50uL rxn

(for AB 7500)

95056-02K

2000 x 50uL rxn

PerfeCta Control Small RNA Primer

Contact your VWR Life Science Specialist

PerfeCta SYBR Green SuperMix for iQ

95053-100

100 x 50uL rxn

95053-500

500 x 50uL rxn

(for Bio-Rad iCycler)

95053-02K

2000 x 50uL rxn

250 x 50uL assays

PerfeCta SYBR Green SuperMix

95054-100

100 x 50uL rxn

95054-500

500 x 50uL rxn



95054-02K

2000 x 50uL rxn

qScript microRNA Quantification System

31

PerfeCta NGS Quantification Kits

Accurate library quantification for next gen sequencing

PerfeCta NGS Quantifications kits use real-time PCR with prediluted standards for the convenient and accurate quantification of NGS libraries for the Ion Torrent or Illumina NGS platforms.

FEATURES AND BENEFITS Accurate and sensitive method for NGS library quantification Stabilized, prediluted standards for convenient use ■ Consistency across a broad range of samples ■ Sensitivity of detection to quantify low concentration libraries ■ Quantification for either Illumina or Ion Torrent ■ ■

Accurate quantification of the number of amplifiable library molecules is the most critical step in the NGS workflow in obtaining high quality read data with next-generation sequencing technologies. The PerfeCta NGS Library Quantification Kit uses real-time PCR to specifically quantify library molecules that possess the appropriate adapter tag at each end. These are the suitable template molecules for either emulsion PCR used for the Ion Torrent platform or Bridge PCR used for Illumina NGS platforms. PerfeCta NGS Quantification kits simplify the library quantification process by providing stabilized, pre-diluted standards, pre-qualified primer sets, and an optimized dilution buffer for your NGS library samples. This minimizes pipetting errors and ensures reproducible and precise qPCR results, even with dilute samples. The robust qPCR performance of PerfeCta SYBR Green SuperMix provides accurate quantification of NGS libraries with varying fragment sizes or GC content. Kits are available to support all major qPCR instrument platforms. Ion Torrent

32

Illumina

The Ion Torrent DNA standard produces a 183-bp amplicon (51.9% GC) using primers that target the “A” and “trP1” adaptor sequences:

The DNA standard for Illumina NGS platforms generates a 426-bp amplicon (48.8% GC). Primer sequences correspond to the “P5” and “P7” primer sequences for Illumina sequencing libraries:

Ion Torrent forward: 5’-CCA TCT CAT CCC TGC GTG TC-3’

Illumina forward primer: 5’ AAT GAT ACG GCG ACC ACC GA 3’

Ion Torrent reverse: 5’-CCT CTC TAT GGG CAG TCG GTG AT-3’

Illumina reverse primer: 5’-CAA GCA GAA GAC GGC ATA CGA-3’

PerfeCta NGS Quantification Kits

A common problem with some NGS library quantification protocols is the use of DNA standards that are too concentrated and generate qPCR data that are outside of the linear dynamic range for many qPCR instruments. Improper baseline settings result in compressions between the highest concentrated DNA standards that in turn give rise to inflated PCR efficiencies and inaccurate library quantification results. The NGS DNA standards supplied with the PerfeCta NGS Library quantification kits have been carefully selected to avoid these artifacts and produce NGS library standard curves with exceptionally high linear regression correlation coefficients.

A 4000

Slope r PCR Eff.

1000

800

-d(RFU)/dT

2000

Tm=82.5 oC

1000

NTC 0.0005 pM 0.005 pM 0.05 pM 0.5 pM 5 pM

3000

RFU

B

-3.438 -0.9998 95.4%

600 400 200 0

0 0

5

10

15

20

25

30

65

35

C

Slope r PCR Eff.

1000

80

85

90

95

Tm=81.5 oC 800

-d(RFU)/dT

RFU

2000

75

D NTC 1E3 copies/µL 1E4 copies/µL 1E5 copies/µL 1E6 copies/µL 1E7 copies/µL

3000

70

Temperature (oC)

Cycle Number

-3.397 -0.9998 97.0%

600

400

200

0 0 0

5

10

15

20

25

30

35

Cycle Number

65

70

75

80

85

90

95

Temperature (oC)

PerfeC ta NGS library Quantification Kit performance data. qPCR amplification of each of the five supplied DNA standards for Illumina NGS libraries (panel A) or Ion Torrent libraries (panel C) were carried out with the supplied primer sets (300 nM final concentration) and PerfeC ta SYBR Green SuperMix in 20-uL reaction volumes on a Bio-Rad CFX-96. Reactions were incubated for 5 min at 95˚C followed by 35 cycles of: 95˚C, 10s; 60˚C, 20s; 45s, 72˚C. Real-time fluorescence data was collected and analyzed at completion of the 72˚C extension step. After completion of PCR, a dissociation (melt) curve was performed to verify amplification of a single specific product (panels B and D).

NGS Library Quantification kit components:

ORDERING INFORMATION

NGS Primer Mix (10 µM ea.) Pre-diluted standard DNAs Dilution Buffer for NGS library samples Bundled with one of the following: PerfeCta SYBR Green SuperMix PerfeCta SYBR Green SuperMix ROX PerfeCta SYBR Green SuperMix Low ROX PerfeCta SYBR Green SuperMix Low ROX

PRODUCT

Quanta Cat. No.

Perfecta NGS Quantification Kit - Ion Torrent

95151-500

Perfecta NGS Quantification Kit - Ion Torrent/ROX

95152-500

Perfecta NGS Quantification Kit - Ion Torrent/Low ROX

95153-500

Perfecta NGS Quantification Kit - Illumina

95154-500

Perfecta NGS Quantification Kit - Illumina/ROX

95155-500

Perfecta NGS Quantification Kit - Illumina/Low ROX

95156-500

PerfeCta NGS Quantification Kits

33

Summary of Limited Label License Statements for Quanta’s PCR, qPCR, and cDNA synthesis kits. Limited Label License (products that contain antibody hot-start Taq)

Limited Label License (HRM)

Licensed to Quanta BioSciences, under U.S. Patent Nos. 5,338,671, 5,587,287, and foreign equivalents for use in research only.

The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under U.S. Patent No. 5,871,908, owned by Evotec Biosystems GmbH and licensed to Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent No. 5,871,908, other than for the amount of product contained herein.

Limited Label License (licensed PCR reagents – contain a non-ionic detergent) Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to expired US Patent No. 5,079,352. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Limited Label License (qPCR products that contain a passive reference dye, ROX or fluorescein) The purchase of this product includes a limited, non-transferable right to use the purchased amount of the product to perform Applied Biosystems’ patented Passive Reference Method for the purchaser’s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. For information about these rights or on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. Limited Label Licenses (SYBR Green qPCR products) This product is provided under an agreement between Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corporation) and Quanta Biosciences, Inc., and the manufacture, use, sale or import of this product is subject to one or more of U.S. Patent Nos. 5,436,134; 5,658,751 and corresponding international equivalents, owned by Molecular Probes. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851 The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein. Limited Label License (qPCR products with dsDNA-binding dye) Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

34

Limited Use Label License (qScript cDNA SuperMix) This product is covered by US patent 7,470,515, US patent 7,638,612 and other patents pending in the United States and Europe. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer. The buyer is not authorized to sell or otherwise transfer this product, any of its components to a third party. The purchase of this product does not authorize the purchaser to use the product or any of its components for manufacture of commercial product. For information on obtaining a license to this product for purposes other than research, contact Licensing Department, Quanta BioSciences, Inc., 202 Perry Parkway, Suite 1, Gaithersburg, MD 20877; Telephone number: 1-800-364-2149. Limited Use Label License (UNG/dUTP-containing products) The use of this product is covered by at least one claim of U.S. Patent No. 7,687,247 owned by Life Technologies Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product, (b) its components, or (c) materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes. Commercial Purposes means any activity for which a party receives or is due to receive consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. The buyer cannot use this product or its components or materials made using this product or its components for therapeutic, diagnostic or prophylactic purposes. Further information on purchasing licenses under the above patents may be obtained by contacting the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008. Email: [email protected]. Limited Label License (AccuStart II, GelTrack, ToughMix and FastMix II products). This product is licensed under US 7,972,828 and corresponding US and foreign patents and patent applications for any use for research and development purposes; the license expressly excludes any use for diagnostic testing or clinical therapeutics in humans or animals. Disclaimer (ToughMix, FastMix II, One-Step RT-qPCR ToughMix – PCR products for applications other than realtime detection with ds-DNA binding dyes that do NOT contain a non-ionic detergent) The use of certain types of fluorogenic probes with 5’ nuclease assays may be covered by U.S. Patent No. 5,538,848, owned by Life Technologies, Corporation. Purchase of this product does not convey rights to practice the methods claimed in U.S. Patent No. 5,538,848, and a license to practice those methods must be obtained from Life Technologies, 850 Lincoln Center Drive, Forest City, California 94404, or by purchase of fluorogenic probes from an authorized source. Use of other hybridization probes may be covered by other patents and specific licenses may be required for each technology which is not conveyed by purchase of this product. Consult your probe provider for patent and license information related to each probe chemistry and their use. Trademarks: Quanta BioSciences, PerfeCta®, FastMix®, FastMixes™ ToughMix®, qScript™, AccuStart™, Extracta™, GelTrack™, AccuMelt™, and AccuFast™ are trademarks of Quanta BioSciences, Inc.. Applied Biosystems, AmpliTaq, Gold, Gold 360, GTXpress, Path-ID™, SuperScript ®, Platinum®, SYBR®, SYTO9, ThermoScript™, OneStep™ ViiA7, ROX ™, and GreenER™ are trademarks of Life Technology Corporation. iScript™, CFX96™, CFX384™, iQ™, MyiQ™, iCycler ®, iCycler, iQ®, Chromo4™, MiniOpticon™ and Opticon® are trademarks of Bio-Rad Laboratories. TaqMan® and LightCycler ® are registered trademarks of Roche Molecular Systems, Inc. Rotor-Gene® QuantiFast, and QuantiTect ® are trademarks of Qiagen. Mastercycler ® is a trademark of eppendorf. Stratagene, MX3000P®, MX3005P™ and MX4000 ® are trademarks of Agilent Corporation. OneStep™ is a trademark of Applied Biosystems. Smartcycler ® is a trademark of Cepheid Pikoreal

Limited Label License (SYTO 9 dye)

Eco is a trademark of Illumina

This product is provided under an agreement between Life Technologies Corporation (Molecular Probes Labeling and Detection Technologies) and Quanta BioSciences, Inc. and the manufacture, use, sale, or import of this product is subject to one or more U.S. Patents and corresponding international equivalents. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; 2) to provide a service, information, or data to an unaffiliated third party for payment; 3) for therapeutic, diagnostic or prophylactic purposes; 4) for resale, whether or not such product or its components are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation (Molecular Probes Labeling and Detection Technologies), Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300, FAX: (541) 335-0354

Quanta BioSciences is licensed for qPCR see www.quantabio.com for details.

ENABLING

PCR

TECHNOLOGIES

Hot-start, Tough Tested, Ultra-Low in Residual E. coli DNA, Stable and Low Foam for Optical Clarity

antibody mediated HS

ultra low e.coli DNA

www.quantabio.com