KAPA2G Robust PCR Kits FAQs

What are the recommended applications for KAPA2G Robust PCR Kits?

  • Amplification of templates with high GC or AT content
  • Amplification of templates containing common PCR inhibitors at levels inhibitory to wild-type Taq
  • Amplification from crude samples, e.g. buccal swabs; cultured mammalian, bacterial or yeast cells (Colony PCR), mouse tail and ear crude lysates
  • Improvement of yield and/or sensitivity in assays where template quality or primer design is problematic

Why is the KAPA2G Robust PCR Kit the preferred product for Robust PCR?
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.

What are the key areas of optimization?

  • Reaction Set-up
    • Amount of starting template: For high quality DNA, use 1–50 ng genomic DNA or ≤1 ng plasmid/lambda DNA per 25 µL reaction for most applications. For crude samples, DNA contaminated with inhibitors, and low quality DNA, determine the optimal template concentration per reaction in a template dilution series PCR.
    • Template quality: High yields of target amplicons should not be expected with degraded DNA, DNA with a low concentration, and/or DNA containing high levels of PCR inhibitors.
    • Enzyme amount: Use 0.5 U KAPA2G Robust per 25 µL reaction for good quality DNA. Use 1 U enzyme per 25 µL reaction for GC-rich amplicons and other difficult samples.
    • Primer and dNTP concentration: Use a final concentration of 0.2 mM of each dNTP and 0.5 µM of each primer.
    • Genomic target length: Fragments in excess of 6 kb have been amplified successfully using KAPA2G Robust. However, success with long fragments is highly dependent on template quality and primer and template characteristics. For robust amplification of long DNA fragments, use KAPA HiFi DNA Polymerase
  • Cycling Parameters
    • Extension time: Use 15–30 sec/kb per cycle. Too long extension times may result in non-specific amplification or smearing, whereas too short extension times may cause low yields.
    • Annealing time: Use 15 seconds per cycle for good quality DNA. This may be reduced to 10 seconds per cycle for slow cyclers or reduced reaction volumes (<25 µL). For difficult samples, annealing times may be increased to 30 seconds per cycle
    • Annealing temperature: Optimal annealing temperature is not only determined by primer and template characteristics, but also by the chemical environment (buffer, additives and sample composition). Start with a known annealing temperature or use 55°C as a first approach. Perform an annealing temperature gradient PCR to determine the annealing temperature that produces the highest yield of specific product.

What are the key areas of optimization if PCR products generated with KAPA2G Robust contain a high background of non-specific amplicons or high molecular weight smears?

  • Reduce the annealing time to 10–15 seconds per cycle.
  • Increase the annealing temperature or determine the optimal annealing temperature in an annealing temperature gradient PCR. Annealing temperatures <55°C are typically not recommended.
  • Reduce the extension time to 15 sec/kb per cycle for amplicons ≤1 kb and 15–30 sec/kb per cycle for 1 kb–3.5 kb amplicons.
  • Reduce the amount of template in the reaction. For high quality DNA, use 1–50 ng genomic DNA or ≤1 ng plasmid/lambda DNA per 25 µL reaction for most applications.
  • Reduce the number of cycles.
  • Reduce the amount of enzyme per reaction.
  • Try a different KAPA2G Robust buffer supplied in the kit.
  • Include 1x KAPA Enhancer 1 in reaction mixes set up with KAPA2G Buffer A or B.
  • Optimize the MgCl2 concentration.
  • Include DMSO in the reaction to a final concentration of 5%–10%.
  • Reduce the primer concentration, but not lower than 0.1 µM of each primer. Do not use low primer concentrations in combination with lower than usual (0.2 mM each) dNTP concentrations.
  • Redesign primers to eliminate inter- or intra-primer interactions or improve specificity.

What is the recommended extension time for KAPA2G Robust DNA Polymerase?
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.

When should I optimize the amount of KAPA2G Robust DNA Polymerase per reaction?
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.

  • Increase the amount of enzyme per reaction in the following cases:
    • For GC-rich amplicons (use 1 unit per 25 µL reaction or equivalent).
    • For crude templates or templates containing inhibitors (use 1-2 units per 25 µL reaction or equivalent).
    • For long fragments, but only if the template copy number is high (use 1 unit per 25 µL reaction or equivalent).
  • Reduce the amount of enzyme per reaction in the following cases:
    • When smearing occurs, particularly during the amplification of long fragments.
    • When the template concentration is low.
    • When a high background of non-specific amplicons is obtained.

Does magnesium need to be added to the PCR reaction with KAPA2G Robust PCR Kit?
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.

How do I optimize a PCR assay to be used routinely with DNA containing inhibitors or crude samples as template?

  • Obtain the target amplicon in a form that allows for the preparation of good quality template DNA if possible, e.g. clone the target fragment into a plasmid. If this is not possible, create an artificial target by cloning the primer sequences into a plasmid in such a way that amplification would yield an amplicon of similar size and GC content as in the authentic target.
  • Purify template DNA from this source, use it to set up and optimize the basic assay parameters (reagent concentrations and cycling parameters), and determine the sensitivity of the assay (minimum number of target copies detectable).
  • Prepare a 10-fold dilution series of the experimental template. Set up duplicate reactions, in which one set is spiked with a known amount of the target amplicon (from an artificial source, as described above). The amount of artificial control target should be 10–100X more copies than the smallest detectable copy number achieved with good quality DNA during assay optimization.
  • The results from such a dilution series experiment will indicate if inhibitory element(s) in the experimental template may be diluted out, whilst remaining within the sensitivity range for the target amplicon. If a suitable artificial control template is not available, reactions may be spiked with template for a different control amplicon and a second primer set included in the reaction, provided that this control is efficiently amplified under the same reaction conditions.
  • If extensive optimization continues to yield a very low concentration of the target amplicon, use the product from this PCR in a second round of amplification. The same or a nested primer pair may be used in the second round. Perform a series of reactions, containing 1 µL of undiluted, 10X diluted, 100X diluted or 1000X diluted product from the first round. Depending on the application, limit the second PCR to 10–30 cycles.

What is the difference between KAPA2G Robust or KAPA2G Robust HotStart?
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.

What are the storage recommendations for KAPA2G Robust PCR Kit?
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).

Which KAPA2G Buffer should I use in my PCR reaction?
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:

  • KAPA2G Buffer A
  • KAPA2G Buffer A + KAPA Enhancer 1
  • KAPA2G Buffer B
  • KAPA2G Buffer B + KAPA Enhancer 1
  • KAPA2G GC Buffer (do not combine with KAPA Enhancer 1)

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:

  • Use KAPA2G Buffer A (with or without KAPA Enhancer 1) for good-quality DNA or for amplicons with a GC content <65%.
  • Use KAPA2G Buffer B (with or without KAPA Enhancer 1) for low-quality DNA, DNA containing anionic inhibitors, crude samples, or longer amplicons.
  • Use KAPA2G GC Buffer for amplicons with a GC content >65%

For GC-rich amplicons resistent to amplification, the following additional buffer/additive combinations may be tried:

  • KAPA2G GC Buffer + 4% DMSO
  • KAPA2G Buffer A + KAPA Enhancer 1 + 5% DMSO
  • For new assays, it is recommended that all five basic buffer/additive combinations be evaluated in parallel to determine which combination produces the highest yield of specific product before further optimization is attempted.

When should I use the KAPA Enhancer 1 in my PCR reaction?
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.

Can I still use KAPA2G Robust if my existing assay requires a specialized buffer?
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.

Is the KAPA2G Robust PCR Kit compatible with PCR additives?
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:

  • Include a final concentration of 1–10% DMSO in reactions performed with KAPA2G Buffer A or B. Reactions performed with KAPA2G GC Buffer may be supplemented with up to 4% DMSO.
  • Include other PCR additives typically used with wild-type Taq (e.g. BSA, glycerol, single-stranded binding protein, PEG, glycine) in the reaction. KAPA2G Robust typically tolerates higher concentrations of additives than wild-type Taq, but the relative advantage and optimal concentration of each additive will have to be determined empirically.
  • KAPA Enhancer 1 has similar properties to betaine. It may be used at a 1X concentration instead of betaine in any assay shown to benefit from the inclusion of betaine. Do not combine KAPA Enhancer 1 and betaine.
  • The inclusion of 5% glycerol or 0.1% Tween 20 in reactions often improves the amplification of longer fragments. However, KAPA HiFi is recommended for the robust amplification of amplicons >3 kb.

Can PCR products generated with the KAPA2G Robust be digested, cloned and sequenced?
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.

Can PCR products generated with KAPA2G Robust PCR Kits be analyzed by dHPLC?
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.

Can I use the KAPA2G Robust PCR Kit for Multiplex PCR?
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.

How do I prepare templates for crude sample PCR using KAPA2G Robust PCR Kits?
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.

  • Place a small amount of the sample to be tested (e.g. a 2 mm leaf punch, 2 mm tissue segment, 2 µL of a liquid sample) in 50 µL 10 mM Tris-HCl, pH 8–8.5 and vortex for 15 seconds.
  • Incubate for 10 minutes at 95°C. After incubation, vortex for 15 seconds.
  • Centrifuge for 1 minute at maximum speed in a benchtop centrifuge to collect debris in the bottom of the tube.
  • Transfer the supernatant to a new microcentrifuge tube and prepare a 5-fold serial dilution (in 10 mM Tris-HCl, pH 8–8.5). Prepare at least 5 dilutions.
  • Use 2.5 µL of each dilution in PCR reactions with KAPA2G Robust or KAPA2G Robust HotStart, using the reaction setup conditions in the User Guide.
  • Start with the following cycling profile: initial denaturation: 3 minutes at 95°C, denaturation: 15 seconds (95°C) per cycle; annealing: 15 seconds (55 – 65°C) per cycle, extension (72°C): 30 sec/kb per cycle. Number of cycles: 35–40.
  • Evaluate KAPA2G Buffer A and KAPA2G Buffer B (with or without KAPA Enhancer 1) in parallel to determine which buffer/additive combination is optimal for the specific sample type. In most cases, Buffer B is preferred for crude sample PCR.
  • For amplicons with high GC content (60–70%), evaluate KAPA2G GC Buffer or KAPA2G Buffer B plus 5% DMSO.
  • Crude sample PCR is not recommended for amplicons >1 kb or amplicons that are difficult to amplify with wild-type Taq when using high-quality DNA as template.
  • Please refer to the application note Mouse Genotyping for specific details on how to use KAPA2G Robust for mouse genotyping with crude mouse ear or tail lysates as template.

What is the best strategy for GC-rich PCR?

  • Use 1 unit enzyme per 25 µL reaction or equivalent.
  • As a first approach, use 1X KAPA2G GC Buffer without any additives.
  • For particularly resistent templates/amplicons, try 1X KAPA2G GC Buffer + 4% DMSO or 1X KAPA 2G Buffer A + 1x KAPA Enhancer 1 + 5% DMSO.
  • Ensure that templates are properly denatured. Use an initial denaturation time of 5–10 minutes at 95°C or pre-denature the template. Denature for 15-30 seconds at 95°C per cycle.
  • Please refer to the application note Routine GC-Rich PCR for more information.

What is the best strategy for E. coli Colony PCR?

  • Use 0.5–1.0 U enzyme per 25 µL reaction or equivalent.
  • Use KAPA2G Buffer B or KAPA2G GC Buffer for GC-rich amplicons. KAPA Enhancer 1 may improve results in some cases.
  • Use primers at a final concentration of 0.5 μM each and dNTPs at a final concentration of 0.2 mM each. A final MgCl2 concentration of 1.5 mM (as present in all KAPA2G buffers at a 1x concentration) should be sufficient for most Colony PCRs. This may be supplemented to a final concentration of 2-5 mM MgCl2 if yields or specificity is low or the specific amplicon is known to require a higher optimal MgCl2 concentration. This includes AT rich templates.
  • Include 1–2 µL of an E. coli colony resuspended in 10-20 µL PCR water, or 1 µL of an overnight culture per 25 µL reaction.
  • Use an initial denaturation time of 5 minutes (95°C) and 10–15 seconds denaturation time (95°C) per cycle.
  • Use an annealing time of 10–15 seconds per cycle.
  • Use an extension time (72°C) of 5 seconds per cycle for amplicons ≤500 bp, or 15 – 30 sec/kb per cycle for amplicons >500 bp.
  • Start with 30 cycles. Depending on yields, this may be reduced to 25 cycles or increased to 35 cycles.
  • Please refer to the application note Colony PCR for more information.

What is the best strategy for yeast Colony PCR?

  • Use 1.0 U enzyme per 25 µL reaction or equivalent.
  • Use KAPA2G Buffer B or KAPA2G GC Buffer for GC-rich amplicons. KAPA Enhancer 1 may improve results in some cases.
  • Use primers at a final concentration of 0.5 μM each and dNTPs at a final concentration of 0.2 mM each. A final MgCl2 concentration of 1.5 mM (as present in all KAPA2G buffers at a 1x concentration) should be sufficient for most Colony PCRs. This may be supplemented to a final concentration of 2-5 mM MgCl2 if yields or specificity is low or the specific amplicon is known to require a higher optimal MgCl2 concentration. This includes AT rich templates.
  • Lyse yeast cells (overnight colonies or cultures) in 0.1 M NaOH or Zymolase for at least 5 minutes at 37°C.
  • Include 2.5 µL of the lysed yeast cell suspension per 25 µL reaction.
  • Use an initial denaturation time of 5 minutes (95°C) and 30 seconds denaturation time (95°C) per cycle.
  • Use an annealing time of 10–15 seconds per cycle.
  • Use an extension time (72°C) of 15 seconds per cycle for amplicons ≤500 bp, or 30 sec/kb per cycle for amplicons >500 bp.
  • Start with 30 cycles. Depending on yields, this may be reduced to 25 cycles or increased to 35 cycles.

Materials

     
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