For which applications should I use KAPA Long Range PCR Kits?
What is the enzyme in the KAPA Long Range PCR Kit?
The KAPA Long Range PCR system is a blend of Taq DNA polymerase and a modified archaeal (Type B) DNA polymerase possessing proofreading capability. This two-enzyme system is designed specifically to support long-range and/or sensitive PCR. Both enzymes possess 5′-3′ polymerase activity, but only Taq possesses double strand dependent 5′-3′ exonuclease activity and only the Type B polymerase possesses 3′-5′ exonuclease (proofreading) activity.
What are the key areas of optimization?
What are the standard cycling conditions for KAPA Long Range and KAPA Long Range HotStart?
For simple PCR (short range/high template concentration), KAPA Long Range Polymerase can be used instead of Taq without modification of the normal protocol. However, as the reaction becomes more difficult, i.e. longer range/lower template concentration, the PCR conditions must be adjusted by increasing the number of cycles, decreasing extension temperature, increasing the amount of enzyme, and using an auto-extend step during the later cycles of PCR. Three examples of typical PCR cycling conditions are described below.
Short targets (up to 8kb) and/or high concentrations of template DNA
Longer targets (5 kb-18 kb) and/or lower concentrations of template DNA
Very long targets (15 kb and larger) and/or lower concentrations of template DNA
What is the recommended extension rate and temperature for KAPA Long Range PCR Kits?
An extension time of 1 minute per 1 kb should be suitable for most PCR assays. For short targets (up to 8 kb) and/or reactions with high concentrations of template DNA an extension temperature of 72 ˚C should be used. For longer targets (5 kb-18 kb) and/or lower concentrations template DNA an extension temperature of 68 ˚C should be used. For very long targets (15 kb and larger) and/or low concentrations of template DNA an auto-extension step and an extension temperature of 68 ˚C should be used.
When should I optimize the amount of enzyme per reaction?
For short targets (up to 8 kb) and/or high concentrations of template DNA and longer targets (5 kb-18 kb) and/or lower concentrations of template DNA, 1.25 U/50 µL should be used. However, for very long targets (15 kb and larger) and/or low concentrations of template DNA 2.5 U/50 µL should be used.
Does magnesium need to be added to the PCR reaction with the KAPA Long Range PCR Kit?
The KAPA Long Range Buffer supplied with the KAPA Long Range PCR Kits does not contain magnesium. However, a separate tube of MgCl2 is included in all kits. We recommend starting with a final concentration of 1.75 mM in your PCR reaction. If this leads to suboptimal results (i.e. smearing and nonspecific products) determine the optimal MgCl2 for your assay by performing an MgCl2 gradient PCR, during which the final MgCl2 concentration in the reaction is increased in increments of 0.5 mM.
What is the difference between KAPA Long Range and KAPA Long Range HotStart?
KAPA Long Range HotStart is comprised of the two-enzyme blend combined with 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 order KAPA Long Range HotStart ReadyMix with dye?
KAPA Long Range HotStart ReadyMix with dye is recommended for routine PCR that 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 conditions for KAPA Long Range PCR Kit?
The recommended temperature for long-term storage of the KAPA Long Range PCR Kit is -20 ˚C. However, kit components or PCR master mixes prepared from them may be stored at 4 ˚C for short-term usage (up to one month).
Can I still use the enzymes supplied in KAPA Long Range PCR Kit if my existing assay requires a specialized buffer?
The buffers supplied in KAPA Long Range and KAPA Long Range HotStart PCR Kits have been optimized specifically for the KAPA Long Range PCR System and it is highly recommended that the buffer supplied in this kit are evaluated as a first approach.
Is the KAPA Long Range PCR Kit compatible with PCR additives?
For GC-rich or other difficult templates or amplicons, DMSO can be added into the PCR reaction at a final concentration of 5%.
Can PCR products generated with KAPA Long Range PCR Kit be digested, cloned, and sequenced?
The KAPA Long Range PCR system is a blend of Taq DNAPolymerase and a modified archaeal (Type B) DNA polymerase possessing proofreading capability. Therefore, the PCR products generated with the KAPA Long Range PCR Kit has similar characteristics as PCR products generated with wild-type Taq or hot start formulations. As such, they are suitable for routine downstream applications such as digestion with restriction endonucleases and sequencing. PCR products generated 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.
Can PCR products generated with KAPA Long Range PCR Kits be analyzed by dHPLC?
Yes. PCR products generated with KAPA Long Range and KAPA Long Range HotStart using the KAPA Long Range Buffer at the recommended final concentrations, do not contain mineral oil, formamide, Proteinase K, BSA, high molecular stabilizers (e.g. PEG), detergents (e.g. SDS, Triton X-100, Tween 20, and Nonidet P40), glycerol, betaine or DMSO at final concentrations exceeding the maximum allowable concentrations for direct analysis using Transgenomic WAVE dHPLC systems.