What are the recommended applications for KAPA2G Fast PCR Kits?
- Standard, end-point PCR assays normally performed with wild-type Taq (or hot-start formulations thereof) – but completed in 20%–70% less time
- High-throughput end-point PCR
- End-point PCR for genetic testing or downstream Sanger sequencing
- Fast Multiplex PCR
What is Fast PCR?
Fast PCR is PCR in which total reaction time is decreased by reducing the duration of one or more of the steps during each cycle of a PCR. Using the KAPA2G Fast PCR Kit, PCR time can be reduced by 20%–70% by simply reducing the extension time in each cycle. Additional timesaving may be achieved by optimizing other cycling parameters (e.g. denaturation and/or annealing time) for your specific assay and thermal cycler.
Why is the KAPA2G Fast PCR Kit the preferred product for Fast PCR?
KAPA2G Fast PCR Kits are unique as they contain the KAPA2G Fast DNA Polymerase, a second-generation enzyme that is capable of synthesizing DNA much faster than Taq and other DNA polymerases. High-performance, high-speed PCR is possible without the need for extensive optimization, special PCR consumables or specialist thermal cyclers. In contrast, competitor kits based on wild-type Taq polymerase are limited by the extension rate of Taq, and timesaving relies primarily on the reduction of denaturation and annealing times. Such “artificially” shortened protocols often result in reduced reaction efficiency, especially with longer amplicons, lower-target copy numbers and faster thermal cyclers.
Can any existing PCR assay be converted to a Fast PCR assay?
- Assays recommended for conversion:
- Any standard, single-amplicon end-point PCR assay provided that reaction volumes do not exceed 25 µL and a sufficient amount of good quality target DNA is used
- Reactions involving the amplification of fragments up to 5 kb from simple (e.g. plasmid or lambda) DNA and up to 3.5 kb from complex (e.g. genomic) DNA
- Assays not recommended for conversion:
- PCR assays that do not work well with wild-type Taq
- PCR assays that have not been optimized
- Primer-template combinations that produce low yields of specific product, even after extensive optimization
- Low-efficiency PCR assays
- Applications such as fingerprinting PCR, mutagenesis PCR or PCR involving the incorporation of nucleotide analogs
What are the key areas of optimization?
- Reaction Set-up
- Amount of starting template: 10–25 ng genomic DNA or 1–10 ng plasmid/lambda DNA per 25 µL reaction is sufficient for most applications.
- Template quality: Successful Fast PCR requires DNA that is not degraded or contaminated with inhibitors. Only use template DNA with an A260/A280 ratio between 1.7 and 2.0.
- Enzyme amount: Use 0.5 U KAPA2G Fast per 25 µL reaction.
- Primer and dNTP concentrations: Use a final concentration of 0.2 mM of each dNTP and 0.5 µM of each primer.
- Genomic target length: Fragments up to 5 kb may be amplified from plasmid or lambda DNA with KAPA2G Fast, but fast amplification of genomic targets >3.5 kb is not recommended.
- Reaction volumes: Do not exceed reaction volumes of 25 µL.
- Cycling Parameters
- Extension time: Use 1 sec per cycle for amplicons ≤1 kb. This may be increased to a maximum of 5 sec per cycle to improve yields. For amplicons >1 kb, use an extension time of 15 sec/kb per cycle. Too long extension times are likely to result in non-specific amplification and/or smearing.
- Annealing time: Use 15 sec per cycle, or 10 sec per cycle for slow cyclers or reduced reaction volumes (<25 µL).
- Annealing temperature: Use an annealing temperature in the range of 55–65°C, or perform an annealing temperature gradient PCR (52–72°C) to determine the optimal annealing temperature for your assay.
What are the recommended extension rates for KAPA2G Fast DNA Polymerase?
The recommended extension rate depends on amplicon length. For amplicons ≤1 kb, use 1 sec extension time per cycle. This may be increased to a maximum of 5 sec per cycle to improve yields. For amplicons >1 kb, use an extension time of 15 sec/kb per cycle. Too long extension times are likely to result in non-specific amplification and/or smearing.
What is the optimal annealing time for Fast PCR?
Annealing times that are too short may lead to low yields or reaction failure, whereas annealing times that are too long are likely to generate non-specific amplicons or smears. Never use more than 15 sec annealing time per cycle. For small reaction volumes (<25 µL) or slow thermal cyclers, this may be reduced to 10 sec per cycle.
What is the optimal annealing temperature for Fast PCR?
The optimal annealing temperature for any PCR assay depends on the specific template-primer combination. Primers for Fast PCR assays should be designed for use at an annealing temperature between 55°C and 65°C. When converting an existing assay to a Fast PCR assay, start with the same annealing temperature as used with wild-type Taq as a first approach. To obtain the highest yield of specific product with KAPA2G Fast PCR Kits, perform an annealing temperature gradient PCR in the range of 52–72°C.
Why is reaction volume important for successful Fast PCR?
Efficient denaturation is important for reaction performance and is dependent on efficient heat transfer to reaction components. Heat transfer becomes less efficient as reaction volume is increased. Since the temperature inside the reaction tube lags behind the temperature of the thermal cycler block – and this lag is bigger on fast cyclers than on slow cyclers – it is important to ensure optimal heat transfer. Always use the smallest reaction volume (always <25 µL), thin-walled PCR tubes or plates that fit the thermal cycler block well, and sufficient denaturation times.
What is the difference between KAPA2G Fast and KAPA2G Fast HotStart?
KAPA2G Fast HotStart is comprised of KAPA2G Fast DNA Polymerase and a proprietary antibody that inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency and sensitivity.
When should I use KAPA2G Fast HotStart ReadyMix?
KAPA2G Fast HotStart ReadyMix is recommended for any Fast PCR assay, except for Fast Multiplex PCR, instead of using the separate components supplied in the KAPA2G Fast HotStart PCR Kits. Also, it is the preferred product for high-throughput Fast PCR.
When should I use KAPA2G Fast ReadyMix with dye?
KAPA2G Fast ReadyMix with dye is recommended for routine PCR that does not require a HotStart formulation and employs agarose gel electrophoresis as the method of analysis. The ReadyMix already contains loading dye, which allows for loading of PCR products on gels directly after PCR.
What are the storage recommendations for KAPA2G Fast PCR Kit?
The recommended temperature for long-term storage of KAPA2G Fast enzymes, KAPA2G Buffer A, 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).
When should I use a KAPA2G Fast HotStart PCR Kit with Mg-free buffer?
Kits with Mg-free buffer are only recommended for Multiplex PCR applications. It is only supplied in combination with the KAPA2G Fast HotStart enzyme. It is identical to Buffer A with the exception that it does not contain Mg.
Can I still use KAPA2G DNA Polymerase if my existing assay requires a specialized buffer?
KAPA2G Buffer A, supplied in KAPA2G Fast and Fast HotStart PCR Kits, is a proprietary buffer designed specifically for Fast PCR. For optimal results with KAPA2G Fast enzymes, it is highly recommended that this buffer be used. KAPA2G Fast enzymes should be compatible with any standard PCR buffer developed for use with wild-type or hot-start Taq, provided that the pH is 8.3 or higher. Optimal performance in specialized buffers is not guaranteed and it is likely that annealing temperature and time, as well as extension time will have to be determined empirically.
Are KAPA2G Fast enzymes compatible with PCR additives?
KAPA2G Fast PCR Kits are not recommended for assays that require additives, as these are typically “difficult”. KAPA2G Robust PCR Kits are recommended instead. However, KAPA2G Fast enzymes may be used for assays performed successfully with wild-type Taq in the presence of DMSO at a final concentration ≤5%.
Can PCR products generated with KAPA2G Fast PCR Kits be digested, cloned and sequenced?
Yes, PCR products generated with KAPA2G Fast enzymes have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-dA-tailed and may be used for TA cloning, or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, purification of PCR products using any standard PCR cleanup kit is recommended.
Can PCR products generated with KAPA2G Fast PCR Kits be analyzed by dHPLC?
Yes. PCR products generated with KAPA2G Fast or KAPA2G Fast HotStart, using KAPA2G Buffer A or M 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. PCR products generated with KAPA2G Fast HotStart ReadyMix must be diluted >2.5 times, or purified using a standard PCR cleanup kit, prior to analysis on Transgenomic WAVE dHPLC systems.
Is Fast Multiplex PCR possible?
Existing Multiplex PCR assays can be converted to Fast Multiplex PCR assays with KAPA2G Fast HotStart. Optimal reaction parameters for Fast Multiplex PCR are slightly different to those recommended for standard, single-amplicon assays. Please refer to the KAPA2G Fast HotStart Multiplex PCR Application Note for full details of reaction conditions and optimization strategies for Fast Multiplex PCR. KAPA2G Fast HotStart PCR Kits with Mg-free buffer is recommended for Multiplex PCR applications, as this facilitates the optimization of Mg concentration.
Do I have to buy a special thermal cycler to do Fast PCR?
KAPA2G Fast PCR Kits may be used for Fast PCR on any conventional (Peltier-based) thermal cycler with a block, together with standard thin-walled PCR tubes or plates. Using this kit, a significant amount of PCR time may be saved irrespective of whether you have a slow or fast cycler. For optimal performance, it is important to know the approximate ramping (heating and cooling) rates of your specific instrument. Total reaction times on fast cyclers are shorter, but the times programmed for each step of the PCR may have to be longer than for a slow cycler to ensure equal performance (yield/sensitivity).
Should I convert to a 2-step profile to save time?
Two-step profiles are only suitable for primer pairs that anneal effectively above 65°C or across the range of 65–72°C and are not recommended for existing primer pairs with sharply defined optimal annealing temperatures <65°C. Converting from a 3-step to a 2-step assay with unsuitable primers is likely to require extensive optimization to reduce reaction time without compromising reaction performance, especially on fast ramping thermal cyclers. A significant amount of time can be saved by converting an existing 3-step assay to a Fast 3-step assay by simply applying the unique, fast extension rates of the KAPA2G Fast DNA Polymerase without extensive optimization or compromising reaction performance.