143 EUROGENTEC 2008 Catalogue > www.eurogentec.com >
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8 PCR and RT kits & reagents Taq DNA polymerases overview
144
Taq DNA polymerases
145
®
GoldStar DNA polymerase RedGoldStar DNA polymerase HotGoldStar DNA polymerase SilverStar DNA polymerase DAp GoldStar® DNA polymerase
PCR Kits Ready-to-use MasterMixes
147 147 148 148 150
151 151
Nucleotides
152
dNTP mix dNTP set dNTP/dUTP mix UNG
152 152 153 153
RT enzymes
153
Mu-MLV reverse transcriptase
153
Modifying enzymes
154
T4 polynucleotide kinase T4 DNA ligase Tth DNA ligase Tth DNA polymerase DNase I Benzonase® Proteinase K
154 155 155 156 156 157 157
Belgiums,UXEMBOURG Eurogentec Headquarters
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5NITED+INGDOM Eurogentec Ltd.
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&RANCE Eurogentec France s.a.s.u.
+33 2 41 73 33 73 +33 2 41 73 10 26 =
[email protected]
N
N
N
N
GoldStar®
HotGoldStar
SilverStar
DAp GoldStar®
'ERMANYs!USTRIA Eurogentec Deutschland GmbH
+49 221 258 94 55 +49 221 258 94 54 =
[email protected]
N
HotGoldStar Mix
Y
Y
Red’y’Gold Mix
Red’y’Star mix
N
GoldStar Mix
®
Y
RedGoldStar
4RACKING DYE
Y
Y
Y
Y
Y
Y
Y
Y
Y
%XONUCLEASE
N
N
N
N
Y
N
N
N
N
0ROOFREADING EXONUCLEASE
Y
Y
Y
Y
Y
Y
Y
Y
Y
hEXTENDASEv ACTIVITY 4!CLONING
Y
Y
N
N
N
N
Y
N
N
(OTSTART ACTIVITY
MasterMix: HotGoldStar, dNTPs, MgCl2, buffer, red dye - extra tube of 25 mM MgCl2
2
2
12
- 2x MasterMix: GoldStar®, dNTPs, MgCl2, buffer, red dye - extra tube of 25 mM MgCl2 - 2x MasterMix: HotGoldStar, dNTPs, MgCl2, buffer - extra tube of 25 mM MgCl2
12
30 on lambda or 10 on gDNA
- 2x MasterMix: GoldStar , dNTPs, MgCl2, buffer - extra tube of 25 mM MgCl2
®
- 10x optibuffer without Mg2+ - 50 mM MgCl2 - 5x HISpec additive
10
2
- 10x buffer without MgCl2 - extra tube of MgCl2 - 10x buffer without MgCl2 - extra tube of MgCl2
12
12
-AXIMUM SIZEKB
- 10x buffer without MgCl2 - extra tube of MgCl2
-10x buffer without MgCl2 - extra tube of MgCl2
3UPPLIEDWITH
Taq polymerase overview
Taq DNA polymerases overview
Hotstart PCR
Hotstart PCR
All PCR Fragment analysis
All PCR Fragment analysis
Cloning GC-rich templates Long amplicon
High yield PCR PCR product arrays
Hotstart PCR Real-Time PCR
All PCR Fragment analysis
All PCR Fragment analysis
!PPLICATION
PK-0073-02R (200 RXNs)
PK-0073-02 (200 RXNs)
PK-0064-02R (200 RXNs)
168.90
168.90
154.30
154.30
448.90
ME-0068-05 (500 U) PK-0064-02 (200 RXNs)
243.50
254.90
75.00 298.90 2988.00
70.70 254.90 2420.00
70.70 254.90 2420.00
EUR
ME-0068-01 (250 U)
ME-0074-05 (500 U)
ME-0073-01 (100 U) ME-0073-05 (500 U) ME-0073-1ML (5000 U)
ME-0064-01 (100 U) ME-0064-05 (500 U) ME-0064-1ML (5000 U)
ME-0065-01 (100 U) ME-0065-05 (500 U) ME-0065-1ML (5000 U)
2EFERENCE
281.50
281.50
257.00
257.00
708.70
383.50
400.60
125.70 502.40 5023.00
111.80 400.60 3829.00
111.80 400.60 3829.00
CHF
112.60
112.60
102.80
102.80
320.70
173.60
182.40
50.80 201.40 2013.00
50.60 182.40 1732.00
50.60 182.40 1732.00
GBP
PCR and RT kits & reagents
8
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145
Taq DNA polymerases How to make your PCR a success? Low specificity, non-specific amplicon or high background in your PCRs?
Difficulties to amplify GC-rich and long amplicons?
Cause
Cause
Non-specific priming at low temperatures during reaction set-up and initial denaturation.
Taq polymerase is unable to read over GC-rich sequences, due to their high Tm and therefore incomplete denaturation.
Solution
Solution
Use of the HotGoldStar hotstart Taq polymerase. The HotGoldStar Taq polymerase is a modified Taq polymerase, which completely lacks any activity below 74 °C. In this way any amplification of DNA using primers, which have hybridized in a non-specific way is avoided. HotGoldStar requires a thermal activation of 10 minutes at 95 °C to reach maximal initial activity. During the PCR process the rest of the activity is released.
Use of DAp GoldStar® (Difficult Applications GoldStar®). DAp GoldStar® is a Taq polymerase supplied with a special buffer, which allows you to amplify GC-rich templates without the usual problems.
Cause Taq polymerase is unable to read to the end of the long fragment.
Cause Solution High amounts of E. coli DNA amplified in the PCR. Use of DAp GoldStar® (Difficult Applications GoldStar®). DAp GoldStar® is a Taq polymerase supplied with a special buffer, which
Solution
allows you to amplify long templates up to 30 kb. Use of the Eurogentec SilverStar, GoldStar®, RedGoldStar, HotGoldStar or DAp GoldStar®. All these enzymes are purified according to special multiple purification protocols minimizing the amount of E. coli DNA trace present in the final Taq polymerase preparation, allowing you to work with minimal backgrounds. 9ecf[j_jeh7 >ej=ebZIjWh
:7f=ebZIjWh
Figure 6.1: Non-specific priming is avoided with HotGoldStar
Figure 6.2: The DAp GoldStar® allows to amplify easily GC-Rich Amplicons
EUROGENTEC 2008 Catalogue > www.eurogentec.com >
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Belgiums,UXEMBOURG Eurogentec Headquarters
+32 4 372 74 00 +32 4 372 75 00 =
[email protected]
5NITED+INGDOM Eurogentec Ltd.
+44 (0) 1794 511 411 +44 (0) 1794 522 417 =
[email protected]
&RANCE Eurogentec France s.a.s.u.
+33 2 41 73 33 73 +33 2 41 73 10 26 =
[email protected]
'ERMANYs!USTRIA Eurogentec Deutschland GmbH
+49 221 258 94 55 +49 221 258 94 54 =
[email protected]
4HE.ETHERLANDS Eurogentec Nederland b.v.
+31 43 352 06 98 +31 43 354 19 65 =
[email protected]
3WITZERLAND Eurogentec s.a. succursale de Genève
FR: +32 4 372 74 00 DE: +49 221 258 94 55 FR: +32 4 372 75 00 DE: +49 221 258 94 54
8
PCR and RT kits & reagents
Low reproducibility between your PCRs?
Low sensitivity and low final yields of your PCRs?
Cause
Cause
Pipetting inaccuracies
Fast heat degradation of the Taq polymerase
Solution Use of the extremely thermostable GoldStar® or HotGoldStar. GoldStar® and HotGoldStar are recombinant Taq polymerases that were isolated from extremely thermostable clones of Thermococcus aquaticus. They keep more than 80 % activity during the first hour of the PCR, compared to 60 % activity for most standard Taq polymerases (see figure 6.5). Figure 6.3: Example of a low reproducibility
Cause
Solution
Low enzyme activity
Use of the GoldStar® Mix, Red’y’Gold Mix, HotGolStar Mix or Red’y’Star Mix ready-to-use PCR MasterMixes to avoid pipetting inaccuracy. All PCR MasterMixes are prepared according to strict guidelines and each batch is extensively quality controlled.
Solution Use of the SilverStar, a highly active Taq polymerase.
9ecf[j_jeh
=ebZIjWh
Cause Omitted to add Taq polymerase to each reaction.
Solution Use of the RedGoldStar, GoldStar® with a red tracking dye. When using this tracking dye it is possible to verify the addition of the RedGoldStar to the reaction mix. In post-PCR analysis the reaction mix can be applied directly on a gel, without addition of any further
Figure 6.4: The use of the more heat-stabile GoldStar® will give higher sensitivity and higher final yields of your PCRs.
dyes. ® GoldStar 20 U/mg
4 °C lyophilized
Reaction conditions
Storage buffer
67 mM K3PO4 (pH 7.5) Haemoglobin (2 %) 25 °C
1 mg/ml in 50 mM Tris-HCl (pH 8.0) 1 mM CaCl2 4 °C
Benzonase® $ESCRIPTION ®
Benzonase
Belgiums,UXEMBOURG Eurogentec Headquarters
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Proteinase K
Size
2EFERENCE
EUR
CHF
GBP
10000 U
ME-0280-10
345.40
579.10
231.70
5NITED+INGDOM Eurogentec Ltd.
+44 (0) 1794 511 411 +44 (0) 1794 522 417 =
[email protected]
&RANCE Eurogentec France s.a.s.u.
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$ESCRIPTION Proteinase K
'ERMANYs!USTRIA Eurogentec Deutschland GmbH
+49 221 258 94 55 +49 221 258 94 54 =
[email protected]
Size
2EFERENCE
EUR
CHF
GBP
100 mg
ME-0420-10
116.70
196.30
72.50
4HE.ETHERLANDS Eurogentec Nederland b.v.
+31 43 352 06 98 +31 43 354 19 65 =
[email protected]
3WITZERLAND Eurogentec s.a. succursale de Genève
FR: +32 4 372 74 00 DE: +49 221 258 94 55 FR: +32 4 372 75 00 DE: +49 221 258 94 54
