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General Protocol

General Protocol

KAPA2G Fast DNA Polymerase is a second-generation (2G) enzyme derived through a process of molecular evolution. The polymerase was engineered for higher processivity and speed, offering significantly faster extension rates than wild-type Taq polymerase. In addition to speed, KAPA2G Fast provides higher yields and sensitivity than competitor enzymes across a broad range of targets.

Apllications

Any existing PCR assay performed efficiently with wild-type Taq polymerase may be converted to a Fast PCR assay with KAPA2G Fast DNA Polymerase. Typically, very little re-optimization of reaction parameters is required. Fast PCR assays with KAPA2G Fast may be performed with any conventional Peltier-based thermocycler and thin-walled PCR tubes or plates.
Conversion to Fast PCR is not recommended for assays that do not yield optimal results with wild-type Taq such as:

  • Amplification of long fragments (>1 kb) from low target copy numbers.
  • PCR assays involving primers that are prone to non-specific amplification (even after reaction optimization).
  • Complex PCR assays, e.g. Multiplex PCRs or assays involving the incorporation of nucleotide analogs.
  • Optimized assays that give low yields of the desired amplicon despite a high target copy number (e.g. amplification from difficult templates or templates containing PCR inhibitors or low sensitivity assays requiring a polymerase blend).

Although it is possible to convert such assays to Fast PCR assays, significant reaction optimization is likely to be required

1.Reaction Setup

Component Final Concentration 25 µl Reaction
PCR Water   Ajuster à 25 µl
Forward Primer (10 μM) 0.5 μM 1.25 μl
Reverse Primer (10 μM) 0.5 μM 1.25 μl
5X KAPA2G Buffer A 1X 5.0 µl
MgCl2 (25 mM) 1.5 mM 0.5 µl
dNTP Mix (10 mM each) 0.2 mM each 0.5 µl
DMSO (for amplicons with a GC content >60%) 5.0 - 7.5% 1.25 - 1.875 µl
Template DNA As required ≤ 100 ng for genomic DNA
≤  10 ng for less complex DNA(e.g. plasmid, lambda)
KAPA2G Fast DNA Polymerase (5 U/µl) 0.5 U/ 25 µl 0.10 µl



2. Cycling Parameters

Step
 Time and Temperature Cycles
Initial denaturation 1-3 min at 95ºC 1
Denaturation 10 sec at 95ºC 25-40
Annealing 10 sec at optimal Th
Extension 1 sec at 72 °C for amplicons <1 kb, 15 sec/kb at 72 °C for >1 – 5 kb amplicons
Final extension 1-10 min/kb at 72ºC 1
Hold 4-10°C 1


3. Products

Description
 Size
 Catalog#
KAPA 2G Fast PCR Kits with dNTP 100 U KK5008
250 U KK5009
KAPA 2G Fast PCR Kits without dNTP 100 U KK5020
250 U KK5021
KAPA 2G Fast readymix + Dye 100 rxn KK5101
500 rxn KK5102
KAPA 2G Fast HotStart PCR Kits with dNTP 100 U KK5530
250 U KK5502
500 U KK5500
KAPA 2G Fast HotStart PCR Kits without dNTP 100 U KK5523
250 U KK5503
500 U KK5501
2 500 U KK5519
KAPA 2G Fast HotStart PCR Kits without dNTP with Mg free buffer 250 U KK5512
500 U KK5510
KAPA 2G Fast HotStart PCR Kits without dNTP without Mg free buffer 250 U KK5513
500 U KK5511
KAPA 2G Fast HotStart Readymix PCR Kits 100 rxn KK5603
500 rxn KK5601
KAPA 2G Fast HotStart Readymix with Dye 100 rxn KK5608
500 rxn KK5609



4. Résultats

Amplification of 5 human gene fragments using KAPA2G Fast HotStart or competitor hot start Taq formulations. Reactions (25 µl) contained 5 ng human genomic DNA and 0.5 units (KAPA2G Fast HotStart or Competitor I) or 0.625 units (Competitor A or Competitor Q) enzyme. For amplicons with a GC content >65%(#2 and 3), 7.5%DMSO was included in reaction mixes. Cycling was performed on an EppendorfMastercyclerepgradient S, using 3-step cycling profiles (35 cycles) with 15 sec denaturation (95ºC) and 15 sec annealing (60ºC) per cycle for all enzymes. Extension (72ºC) was 1 sec per cycle for KAPA2G Fast HotStart and 60 sec per cycle for competitor enzymes. Initial denaturation/enzyme re-activation and final extension times used for each enzyme were as per the manufacturers recommendations. The total reaction time for each enzyme is as indicated.