KAPA2G Robust PCR Kits are unique as they contain the KAPA2G Robust DNA Polymerase, a second-generation enzyme that was engineered through a process of directed evolution. The novel amino acid mutations in KAPA2G Robust DNA Polymerase offer higher processivity and specific activity, which translates to robust performance across a wide range of AT- and GC-rich amplicons. In addition, the KAPA2G Robust DNA Polymerase exhibits improved tolerance to common PCR inhibitors (e.g. salts, urea, SDS and ethanol) when compared to wild-type Taq or so-called “robust” blends of thermostable polymerases.
An extension time of 15 sec/kb per cycle is suitable for most PCR assays, and may even be reduced to 5–10 sec/kb per cycle when a high concentration of good quality template is used. However, the extension time may be increased to 30 sec/kb per cycle for crude samples, GC-rich amplicons or templates, or other difficult assays. Reduce the extension time to 15 sec/kb per cycle if smearing occurs or if a high background of non-specific amplicons is obtained.
0.5 units of KAPA2G Robust DNA Polymerase per 25 µL reaction (or proportionally more or less for larger or smaller reaction volumes) is sufficient for most standard PCR applications.
All three KAPA2G Buffers are 5X buffers that include MgCl2 at a 1X concentration of 1.5 mM. Additional MgCl2 (25 mM) is supplied in all kits for assays that require additional MgCl2 or optimization of the final MgCl2 concentration.
KAPA2G Robust HotStart is an antibody-mediated hot-start formulation of KAPA2G Robust DNA Polymerase. For workflows that require room temperature setup, the HotStart formulation must be used. The non-hot-start and hot-start formulations should have similar results in most assays.
The recommended temperature for long-term storage of KAPA2G Robust enzymes, KAPA2G Buffers A, B and GC Buffer, KAPA Enhancer 1, dNTPs and MgCl2 is -20°C. However, these kit components or PCR master mixes prepared from them may be stored at 4°C for short-term usage (up to one month).
KAPA2G Robust enzymes are supplied with three distinct reaction buffers, and the proprietary additive, KAPA Enhancer 1. This offers five buffer/additive combinations for optimization:
It is impossible to predict which buffer/additive combination will yield the best results for a specific primer-template combination or template type. However, the following guidelines may be used as a starting point:
For GC-rich amplicons resistent to amplification, the following additional buffer/additive combinations may be tried:
KAPA Enhancer 1 is a proprietary PCR additive (DNA destabilizer) that improves reaction efficiency and specificity for some, but not all, primer-template combinations. For problematic assays, first try KAPA2G Buffer A or B, with or without 1X KAPA Enhancer 1 before further optimization is attempted. Do not combine the GC buffer and KAPA Enhancer 1.
The buffers supplied in KAPA2G Robust and KAPA2G Robust HotStart PCR Kits have been developed specifically for the novel KAPA2G Robust DNA Polymerase and it is highly recommended that the buffer/additive combinations supplied in the kit are evaluated as a first approach. KAPA2G Robust enzymes should be compatible with any PCR buffer developed for use with wild-type or hot-start Taq, provided that the pH is 8.3 or higher. When using a custom buffer, reaction parameters (e.g. enzyme, template and MgCl2 concentrations and annealing temperature) may require optimization.
KAPA2G Robust buffers have been formulated for optimal enzyme performance under a wide variety of reaction conditions and with diverse templates and amplicon types, and additional additives should not be required for the majority of applications. If a standard reaction (without any additives) does not yield satisfactory results, always first try 1X KAPA Enhancer 1 (in combination with KAPA2G Buffer A or B) or KAPA2G GC Buffer to improve results. If this still does not work, the following strategies may be explored:
PCR products generated with KAPA2G Robust have the same characteristics as PCR products generated with wild-type Taq or hot-start formulations thereof, and are suitable for routine downstream applications such as digestion with restriction endonucleases and sequencing. PCR products generated with the KAPA2G Robust are 3′-dA-tailed and may be used for TA cloning, or may be blunt-ended or digested with restriction endonucleases prior to cloning. For best results, purification of PCR products using any standard PCR cleanup kit is recommended.
Yes. PCR products generated with KAPA2G Robust or KAPA2G Robust HotStart, using KAPA2G Buffer A (with or without KAPA Enhancer 1), KAPA2G Buffer B (with or without KAPA Enhancer 1), or KAPA2G GC Buffer at the recommended final concentrations do not contain mineral oil, formamide, Proteinase K, BSA, high molecular weight stabilizers (e.g. PEG), detergents (e.g. SDS, Triton X-100, Tween 20, Nonidet-P40), glycerol, betaine or DMSO at final concentrations exceeding the maximum allowable concentrations for direct analysis using Transgenomic WAVE dHPLC systems.
KAPA2G Fast HotStart is the recommended product for Multiplex PCR (when good quality template DNA is available), even if reducing reaction times is not a priority. Please refer to the Application Note: Multiplex PCR for more information. PCR from crude samples is challenging, as amplification efficiencies in Crude Sample PCR are typically lower than those obtained with good-quality, purified DNA as template. KAPA2G Robust HotStart may be evaluated for low-complexity Multiplex PCR assays (2–3 primer sets, amplicons <2 kb) from difficult-to-amplify, crudely extracted, or poor-quality template DNA, in cases where wild-type Taq yields poor results. Optimization of reaction is likely to be required.
Crude sample PCR is a challenging application and it is difficult to predict which amplicons can be successfully amplified from which crude sample types. The protocol given below is a starting point for the preparation of crude templates and crude sample PCR using KAPA2G Robust PCR Kits.
Please refer to the application note Colony PCR for more information.