7. Can I store KAPA2G Fast PCR Kits at room temperature or 4 °C?
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 a month). Don’t worry if you’ve left any component of the kit on your bench overnight or over the weekend – it will still work fine (but don’t make a habit of it!)

8. What is Fast PCR?
Fast PCR is PCR in which total reaction time is reduced 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 time savings may be achieved by optimizing other cycling parameters (e.g. denaturation and/or annealing time) for your specific assay and thermocycler.

9. Why is the KAPA2G Fast PCR Kit the preferred product for Fast PCR?
KAPA2G Fast PCR Kits are unique 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. This means that reaction times can be reduced significantly by simply reducing the extension time in each cycle. With KAPA2G Fast PCR Kits, high performance, high speed PCR is possible without the need for extensive optimization, special PCR consumables or specialist thermocyclers. In contrast, competitor kits based on wild-type Taq polymerase are limited by the extension rate of Taq, and timesaving rely 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 thermocyclers.

10. Can any existing PCR assay be converted to a Fast PCR assay?
Any standard, single-amplicon end-point PCR assay may be converted to a Fast assay, provided that reaction volumes do not exceed 25 µl and a sufficient amount of good quality target DNA is used. The KAPA2G Fast PCR Kit is validated for Fast 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. Fast PCR is not recommended for 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, or advanced PCR applications such as fingerprinting PCR, mutagenesis PCR or PCR involving the incorporation of nucleotide analogs.

11. 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 Kits.

12. Do I have to buy a special thermocycler to do Fast PCR?
KAPA2G Fast PCR Kits may be used for Fast PCR on any conventional (Peltier-based) thermocycler 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). This is because slow cyclers offer “extra time” that contributes to denaturation and annealing because they take longer to heat and cool the block between consecutive steps and cycles.

13. 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.

14. What is the optimal annealing time for Fast PCR?
Annealing time is a critical parameter in successful Fast PCR using KAPA2G Fast enzymes. Annealing times that are too short may lead to low yields or reaction failure, whereas too annealing times that are too long, is 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 thermocyclers, this may be reduced to 10 sec per cycle.

15. 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 and 65 °C. KAPA2G Buffer A, the optimized buffer for Fast PCR, is designed to facilitate specific primer annealing over a wider range of annealing temperatures than other PCR buffers. 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.

16. Should I convert to a 2-step profile to save time?
2-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 thermocyclers. 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.

17. 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 thermocycler 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 thermocycler block well, and sufficient denaturation times. The above is particularly important for some templates, e.g. those with a high GC content or stable secondary structure, which are more difficult to denature.

18. What is the optimal Mg concentration for Fast PCR?
The optimal Mg concentration for any PCR assay depends on the specific combination of template and primers. A final Mg concentration of 1.5 mM is sufficient for the majority of Fast PCR assays. However, if you are converting an existing assay that requires >1.5 mM Mg to a Fast assay, it is important that the same final concentration of Mg is used.

19. 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%.

20. 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 is 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.

21. 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.

22. Can PCR products generated with KAPA2G Fast PCR Kits be analysed 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.

23. What is the loading dye contained in KAPA2G Fast ReadyMix with dye?