KAPA Long Range PCR Kits FAQs

For which applications should I use KAPA Long Range PCR Kits?

  • PCR amplification of long targets and/or PCR using low amounts of template DNA.
  • Standard short- and mid-range PCR amplification.
  • Amplification of PCR products to be used for ligation into 3′-T-overhang cloning vectors.

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?

  • Reaction Set-up
    • Template concentration: Successful long-range PCR can be accomplished using low concentrations of template DNA. Use <100 ng for complex targets (e.g. human genomic DNA) and <1 ng for simple targets (e.g. plasmid or lambda DNA).
    • Template quality: Performance of this kit is heavily dependent upon the quality of the DNA template. Damaged template will detract from the performance, and is particularly acute if target is long and/or template DNA is supplied in low amounts.
    • Enzyme amount: Use 1.25 U/50 µL reaction for short targets (up to 8 kb) and/or high concentrations of template DNA and for longer targets (5 kb-18 kb) and/or lower concentrations of template. For very long targets (15 kb and larger) and/or low concentrations of template DNA 2.5 U/50 µL should be used. For GC-rich and other difficult templates, higher enzyme concentrations (up to 5 U per 50 µL reaction) may be required.
    • Primer quality and concentration: Performance of the kit is heavily dependent upon the nature of the primer pair. Poorly designed primers will detract from the performance and is particularly acute if the target is long and/or template DNA concentration is low. Care should be taken to optimize primer design to eliminate non-specific priming sites or secondary structure formation. Analyze primer stocks spectrophotometrically to confirm the purity and concentration of primers prior to use in KAPA Long Range reactions. Start with a final concentration of 0.5 uM. Concentrations can be altered to optimize the reaction. Always dilute and store primers in a buffered environment (e.g. 10 mM Tris-HCl, pH 7.5–8.5) instead of water.
    • dNTP concentration: Start with a dNTP concentration of 0.3 mM of each dNTP. Concentrations can be altered to optimize the reaction.
    • Magnesium concentration: MgCl2 is not included in the KAPA Long Range Buffer. Start with a concentration of 1.75 mM. High template concentrations typically used in long-range PCR chelate Mg2+, such that high template concentrations require higher MgCl2 concentrations. To determine the optimal MgCl2 for an assay, perform an MgCl2 gradient PCR, during which the final MgCl2 concentration in the reaction is increased in increments of 0.5 mM.
       
  • Cycling Parameters
    • Initial denaturation time: 3 min is recommended for most assays. For GC-rich targets (>65% GC), 5 min may be used.
    • Denaturation time: Denaturation time should be increased from 15 sec for slow-block instruments (<3˚C/sec ramp rate) to 25 sec for fast-block instruments (>3˚C/sec ramp rate).
    • Extension time and temperature: Use 1 min per kb for extension time. For short targets and/or high concentrations of template extend at 72˚C. For longer targets and/or low concentrations of template extend at 68˚C.
    • Annealing temperature: Optimal annealing temperature is not only determined by primer and template characteristics, but also by the chemical environment. Start with an annealing temperature (found with a basic Tm calculator) – 5˚C as a first approach. Perform an annealing temperature gradient PCR to determine the annealing temperature that produces the highest yield of specific product.
    • Cycle number: A higher number of cycles may be necessary for assays requiring higher sensitivity, while lower cycle numbers can be used if the template copy number is high. If high fidelity is required, keep the number of cycles as low as possible. For short targets and/or high concentrations of template start with 25 cycles and for longer targets start with 35 cycles. For very long targets include an auto-extension step.

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

  • Initial denaturation: 3 min at 94˚C
  • Denaturation: 15–25 sec at 94˚C (15 sec for slow-block instruments, 25 sec for fast-block instruments)
  • Annealing: 15 sec at Tm–5˚C
  • Extension: 1 min/kb at 72˚C
  • Final extension: 1 min/kb at 72˚C (Only required if fragments are to be –TA cloned)
  • Number of cycles: 25

Longer targets (5 kb-18 kb) and/or lower concentrations of template DNA

  • Initial denaturation: 3 min at 94˚C
  • Denaturation: 15–25 sec at 94˚C (15 sec for slow-block instruments, 25 sec for fast-block instruments)
  • Annealing: 15 sec at Tm–5˚C
  • Extension: 1 min/kb at 68˚C
  • Final extension: 1 min/kb at 72˚C (Only required if fragments are to be –TA cloned)
  • Number of cycles: 35

Very long targets (15 kb and larger) and/or lower concentrations of template DNA

  • Initial denaturation: 3 min at 94˚C
  • Number of cycles: 10
    • Denaturation: 15–25 sec at 94˚C (15 sec for slow-block instruments, 25 sec for fast-block instruments)
    • Annealing: 15 sec at Tm–5˚C
    • Extension: 1 min/kb at 68˚C
  • Number of cycles: 25
    • Denaturation: 15–25 sec at 94˚C (15 sec for slow-block instruments, 25 sec for fast-block instruments)
    • Annealing: 15 sec at Tm–5˚C
    • Extension: 1 min/kb + 20 sec per cycle at 68°C
  • Final Extension: 1 min/kb at 72˚C (Only required if fragments are to be –TA cloned)

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.

Materials

     
Related Links