KAPA HiFi PCR Kits

Evolved for extreme fidelity, robustness, and low-bias amplification of difficult templates.

KAPA HiFi DNA Polymerase is a novel, single-enzyme system that exhibits industry-leading performance when compared with other high fidelity polymerases and polymerase blends. KAPA HiFi has been engineered to have an increased affinity for DNA without the need for accessory protein domains. The intrinsic high processivity of KAPA HiFi results in significant improvements to yield, sensitivity, speed, target length, and the ability to amplify difficult templates (e.g AT- and GC-rich). The error rate of KAPA HiFi PCR Kits is 100X lower than wild-type Taq DNA polymerase.

Amplifying NGS libraries? Go to our Library Amplification page.


For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

The error rate of KAPA HiFi is 100x lower than Taq polymerase, 40x lower than polymerase blends such as Platinum® Taq High Fidelity, 3x lower than Pfu Ultra, and 2x lower than Phusion. Data on file.

Achieve the highest fidelity

  • Increased processivity, strong proofreading activity, and optimized buffer system result in superior accuracy
  • The error rate of KAPA HiFi is 100X lower than Taq, 40X lower than polymerase blends, 3X lower than Pfu Ultra and 2X lower than Phusion
Amplification of GC-rich DNA fragments. Amplification of different types of GC-rich DNA fragments using KAPA HiFi HotStart (top panel), a competitor engineered proofreading DNA polymerase containing a dsDNA-binding domain (hot start formulation, middle panel) and wild-type Pfu (bottom panel). Amplicon GC content increases from left (yellow) to right (red). Competitor reactions were performed in according to manufacturers’ instructions and were performed in GC Buffer (engineered competitor), or contained DMSO at a final concentration of 5% (Pfu). All reactions contained 25 ng human genomic DNA as template. Data on file.

Improve performance on GC- and AT-rich templates

  • Achieve higher success rates across targets up to 84% GC content
  • Higher yields when amplifying AT-rich templates

Amplify longer targets with greater sensitivity 

  • High-fidelity PCR of long and complex genomic templates
  • Improved sensitivity
  • High speed allows significantly shorter reaction times for long range PCR

Related Products

Amplifying NGS libraries? Check out these Kapa NGS products to improve your workflow and results:

Sample QC

KAPA hgDNA Quantification and QC Kit

Library Amplification

KAPA Library Amplification Kits

Library Preparation

KAPA Hyper Prep Kits

Library Quantification

KAPA Library Quantification Kits

Applications
  • High-fidelity PCR
  • Amplification of DNA fragments for cloning and protein expression or genomic characterization
  • Site-directed mutagenesis
  • Next-generation sequencing library amplification
Kit Specifications and Contents / Storage

Store kits for 18 months at -20°C.

Kits include KAPA HiFi or KAPA HiFi HotStart DNA Polymerase (1 U/µL), Fidelity Buffer (5X), GC Buffer (5X), high-quality dNTPs (10 mM each) and MgCl2 (25 mM). Both buffers contain MgCl2 at a final concentration of 2 mM.

KAPA HiFi HotStart is also available in KAPA HiFi HotStart ReadyMix (2X), which contains DNA Polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM).

Specifications

Spec
Description
Starting Material
DNA, cDNA, plasmid DNA
Input Amount
<25 ng for gDNA or <1 ng of plasmid DNA

Contents

Component
KAPA HiFi Standard or HotStart DNA Polymerase (1 U/µL)
5X KAPA HiFi Fidelity Buffer
5X KAPA HiFi GC Buffer
MgCl2 (25 mM)
dNTP Mix (10 mM each)
FAQs
For which applications should I use KAPA HiFi PCR Kits?

  • High-fidelity amplification of DNA to be cloned for sequencing, mutation analysis, or protein expression
  • Site-directed mutagenesis
  • High-performance sample enrichment for next-generation sequencing applications
  • High-fidelity amplification of GC-rich and other difficult templates

Why is KAPA HiFi DNA Polymerase the preferred enzyme for high-fidelity PCR?

KAPA HiFi DNA Polymerase is a novel, single-enzyme system that has been engineered for high fidelity and increased processivity. The intrinsic high processivity results in significant improvements to yield, sensitivity, speed, target length, and the ability to amplify difficult templates. Furthermore, KAPA HiFi DNA Polymerase possesses strong 3’–5′ exonuclease (or proofreading) activity and has the lowest published error rate (i.e. highest fidelity) of all commercially available B-family DNA polymerases.

What are the key areas of optimization?

For recommendations on how to optimize KAPA HiFi for amplification of next generation sequencing libraries, please see the library amplification FAQ.

Reaction Set-up

  • Amount of starting template: Too much template is likely to lead to non-specific amplification. Use ≤25 ng genomic DNA or ≤1 ng plasmid/lambda DNA per 25 µL reaction.
  • Enzyme amount: Use 0.5 U enzyme per 25 µL reaction.
  • Primer concentration: Use 0.3 uM of each primer. Lower primer concentrations are likely to result in low yields or smearing. Higher primer concentrations will increase primer-dimer formation and non-specific amplification.
  • dNTP:
    • Use a final concentration of 0.3 mM of each dNTP (1.2 mM total dNTPs).
    • B-family (proofreading) DNA polymerases are more sensitive than A-family DNA polymerases (e.g. Taq) to dUTP poisoning. Only use high quality dNTPs supplied by KAPA Biosystems with KAPA HiFi enzymes.

Cycling Parameters

  • Denaturation: KAPA HiFi buffers have a significantly higher salt concentration that most other PCR buffers. To ensure adequate template denaturation, always denature for 20 seconds at 98°C in each PCR cycle.
  • Extension time: For amplicons ≤1 kb, use 15–30 seconds per cycle. For amplicons between 1 and 10 kb, use 30 sec/kb extension time per cycle. For amplicons >10 kb, use 30–60 sec/kb per cycle.
  • Annealing temperature: Due to the high salt concentration of KAPA HiFi Buffers, optimal annealing temperatures are typically higher than they would be for the same primers using a different enzyme system. Annealing temperatures <60°C are not recommended, as they lead to non-specific amplification. Start with an annealing temperature of 65°C when using KAPA HiFi Fidelity Buffer, or 60°C when using KAPA HiFi GC Buffer. If non-specific products are obtained, determine the optimal annealing temperature in an annealing temperature gradient PCR (60–78°C). Because of high annealing temperatures, 2-step protocols with a combined annealing/extension step at 68–78°C may be used.

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

  • Increase the annealing temperature (Ta): It is recommended that an annealing temperature gradient PCR be performed to determine the optimal Ta; especially in the case of primer sets that have a propensity to produce smearing and/or non-specific products. Certain primer sets might require a combined annealing/extension temperature in the range of 75-78°C, especially when a complex genomic template is used.
  • Decrease the annealing and/or extension time: Initially, an annealing time of 15 seconds per cycle and an extension time of 30 sec/kb per cycle are recommended. If smearing or non-specific amplification occurs, a reduction in the annealing time may improve results, particularly on slow-ramping (older) thermal cyclers. If smearing or non-specific amplification products of a higher molecular weight than the target amplicon still occur after optimization of the annealing temperature and time, the extension time should be reduced to 15 sec/kb per cycle.
  • Decrease the enzyme concentration: 0.5 U of KAPA HiFi HotStart enzyme per 25 µL reaction should be optimal for most assays. However, if smearing still occurs after the annealing temperature and annealing and extension times have been optimized, the amount of enzyme may be reduced.
  • Optimize the MgCl2 concentration: Suboptimal MgCl2 concentrations, especially excess MgCl2, may cause smearing and non-specific products. Typically, the 2 mM MgCl2 included in KAPA HiFi Buffers at a 1X concentration is sufficient for most applications. Increasing the MgCl2 concentration may improve results in some assays and is usually required when a high concentration of DNA is used to generate fragments for cloning. To determine the optimal MgCl2 concentration 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.
  • Include PCR additives or enhancers in the reaction: Certain compounds, e.g. DMSO and betaine, are known to enhance the specificity and yield of problematic PCR reactions and can be included in the reaction at various concentrations. KAPA Enhancer 1, a proprietary additive supplied with KAPA2G Robust PCR Kits is recommended for use in problematic KAPA HiFi assays. Please note that PCR additives/enhancers may reduce the fidelity of KAPA HiFi.
  • Check primer concentration and quality: Analyze primer stocks spectrophotometrically to confirm the purity and concentration of primers prior to use in KAPA HiFi reactions. Set reactions up with a final concentration of 0.3 µM of each primer. This should be sufficient for most applications; however, in rare cases the optimal primer concentration may have to be determined empirically. Always dilute and store primers in a buffered environment (e.g. 10 mM Tris-HC, pH 7.5–8.5) instead of in water. If KAPA HiFi yields good results with one primer pair but not another using the same template, the sequences of the nonfunctional primers may have to be rechecked to eliminate non-specific priming sites, or secondary structure formation.
  • Check template concentration and quality: Verify the integrity of template DNA by agarose gel electrophoresis and confirm the concentration and purity by spectrophotometric analysis. Use a control primer set to ensure that amplification is possible from the specific template. Perform a dilution series of template down to approximately 200 copies as inhibitors are sometimes present in a DNA preparation, which prevent amplification.
  • Use KAPA HiFi GC Buffer instead of KAPA HiFi Fidelity Buffer: The GC Buffer may improve specificity and/or yield in troublesome assays. Re-optimization of cycling parameters (especially annealing temperature) might be needed when switching between buffers.

What are the standard cycling conditions for KAPA HiFi and KAPA HiFi HotStart?

For amplification of next generation sequencing libraries, please see the Library Amplification FAQ.

3-step cycling profile (for optimal Ta in the range of 60–70°C):

  • Initial denaturation: 2–5 minutes at 95°C
  • Denaturation: 20 seconds at 98°C
  • Annealing: 15 seconds at optimal Ta (60–70°C)
  • Extension: 15–30 sec/kb at 72°C
  • Final extension: 1–5 minutes at 72°C
  • Number of cycles: 15–35

2-step cycling profile (for optimal Ta in the range of 68–78°C):

  • Initial denaturation: 2–5 minutes at 95°C
  • Denaturation: 20 seconds at 98°C
  • Annealing/extension: 20–45 sec/kb at 68–78°C
  • Final extension: 1–5 minutes at 72°C
  • Number of cycles: 15–35

What is the difference between KAPA HiFi and KAPA HiFi HotStart?

KAPA HiFi HotStart is comprised of KAPA HiFi 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.

What are the storage recommendations for KAPA HiFi PCR Kit?

The recommended temperature for long-term storage of KAPA HiFi enzymes, KAPA HiFi Fidelity and GC Buffer, 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 KAPA HiFi buffer should I use in my PCR reaction?

KAPA HiFi and KAPA HiFi HotStart are supplied with two 5X reaction buffers, KAPA HiFi Fidelity Buffer and KAPA HiFi GC Buffer. KAPA HiFi Fidelity Buffer is recommended as the first approach for most applications.

The GC Buffer should be used:

  • for targets with a high GC content (>65%) or stable secondary structure, and
  • to improve the yield and/or specificity of the target amplicon.

Both buffers contain MgCl2 at a 1X concentration of 2.0 mM. Please note the following important differences between the two buffers:

  • The Fidelity Buffer will produce a 2-fold lower error rate than the GC Buffer in the same assay. The Fidelity Buffer is therefore recommended for all applications where the highest fidelity is required, except for amplicons that fail to amplify in this buffer.
  • Optimal annealing temperatures are likely to be lower in the GC Buffer than the Fidelity Buffer due to the presence of DNA destabilizers in the GC Buffer.

Can I still use KAPA HiFi DNA Polymerase if my existing assay requires a specialized buffer?

KAPA HiFi Buffers are unique and have been designed specifically for KAPA HiFi DNA Polymerase. KAPA HiFi and KAPA HiFi HotStart cannot be used in buffers designed for use with other B-family DNA polymerases.

Does KAPA HiFi enzyme produce blunt ends or fragments with 3'-dA-overhangs?

KAPA HiFi enzymes produce blunt-ended PCR products due to the 3’–5′ exonuclease activity that removes 3′-dA overhangs necessary for TA cloning. PCR products generated with KAPA HiFi enzymes are suitable for cloning using any commercially available blunt-end cloning kit. KAPA HiFi PCR products can also be prepared for cloning into TA vectors as follows:

  • Purify the PCR product (spin columns are recommended) to fully remove the KAPA HiFi enzyme.
  • Set up a 50 µL A-tailing reaction containing the following: purified PCR product, 1 µL 10 mM dATP, 1 U (0.2 µL) KAPA Taq DNA Polymerase, 5 µL 10 x KAPA Taq Buffer A or B, and water up to 50 µL.
  • Incubate for 10 minutes at 72ºC to obtain a 3′-dA-tailed KAPA HiFi PCR product.

Can PCR products generated with KAPA HiFi PCR Kits be analyzed by dHPLC?

  • PCR products generated with KAPA HiFi enzyme and KAPA HiFi Fidelity 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.
  • PCR products generated with KAPA HiFi enzyme and KAPA HiFi GC Buffer must be diluted >2.5 times, or purified using a standard PCR cleanup kit, prior to analysis on Transgenomic WAVE dHPLC systems.

How can I ensure the highest possible fidelity (lowest error rate) in a PCR?

The error rate achieved in a PCR assay is dependent on characteristics of the DNA Polymerase, the chemical environment (e.g. buffer composition and Mg concentration), DNA template quality and number of PCR cycles. To ensure the highest fidelity in a particular assay:

  • Use as much template as possible. 10 ng of plasmid/lambda DNA or 100 ng of genomic DNA is a good starting point. Please note that larger amounts of template may require extra MgCl2 in the PCR reaction. Non-specific amplification associated with high template concentration may be controlled by reducing the number of cycles.
  • Use as few PCR cycles as possible to obtain an adequate yield of the target amplicon for downstream applications. 25 cycles can be used as a good starting point, however for next generation sequencing libraries please see the library amplification FAQ for recommendations. Excessive cycling will lead to an accumulation of errors.
  • Use high quality template. Always dilute and store DNA templates in a buffered solution, e.g. 10 mM Tris pH 8.0–8.5 (with or without 1 mM EDTA).
  • Minimize or eliminate the exposure of the PCR product to UV light.

Can KAPA HiFi DNA Polymerase incorporate dUTP?

Like all B-family polymerases, KAPA HiFi DNA Polymerase is sensitive to dUTP poisoning and unable to extend past a dUTP present in a template strand. To minimize the negative impact of dUTP in KAPA HiFi reactions, always handle template DNA carefully and use the minimum number of cycles. Only use the high quality dNTPs supplied in KAPA HiFi PCR Kits. For applications such as bisulfite libraries with a dUTPs incorporated in the template, KAPA HiFi Uracil + may be used.

Ordering

Kits include KAPA HiFi or KAPA HiFi HotStart DNA Polymerase (1 U/µL), Fidelity Buffer (5X), GC Buffer (5X), high-quality dNTPs (10 mM each) and MgCl2 (25 mM). Both buffers contain MgCl2 at a final concentration of 2 mM. KAPA HiFi HotStart is also available in KAPA HiFi HotStart ReadyMix (2X), which contains DNA Polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM).

Component

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK2502
07958897001
HotStart PCR Kit, with dNTPs
250 U
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KK2501
07958889001
HotStart PCR Kit, with dNTPs
100 U
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KK2102
07958846001
With dNTPs
250 U
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KK2101
07958838001
With dNTPs
100 U
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ReadyMix

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK2602
07958935001
HotStart ReadyMix, 500 x 25 µL reactions
6.25 mL
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KK2601
07958927001
HotStart ReadyMix, 100 x 25 µL reactions
1.25 mL
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