Technical Data Sheet

KAPA HiFi PCR Kit Kapa/Roche Kit Codes and Components

KAPA HiFi DNA Polymerase (1 U/µL) 20 µL

KR0368 – v13.19
KK2103 KAPA HiFi Fidelity Buffer (5X) 1.5 mL
07958854001 KAPA HiFi GC Buffer (5X) 1.5 mL
This Technical Data Sheet provides product information (20 U) MgCl2 (25 mM) 1.6 mL
and a detailed protocol for the KAPA HiFi PCR Kit. KAPA dNTP Mix (10 mM each) 40 µL
This document applies to the following kits:
07958854001, 07958838001 and 07958846001. KAPA HiFi DNA Polymerase (1 U/µL) 100 µL
KK2101 KAPA HiFi Fidelity Buffer (5X) 1.5 mL
07958838001 KAPA HiFi GC Buffer (5X) 1.5 mL
Contents (100 U) MgCl2 (25 mM) 1.6 mL
KAPA dNTP Mix (10 mM each) 160 µL
Product Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Product Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
KAPA HiFi DNA Polymerase (1 U/µL) 250 µL
Product Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 KK2102 KAPA HiFi Fidelity Buffer (5X) 1.5 mL
07958846001 KAPA HiFi GC Buffer (5X) 1.5 mL
Important Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 (250 U) MgCl2 (25 mM) 1.6 mL
Standard PCR Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . 4 KAPA dNTP Mix (10 mM each) 300 µL

Appendix A - Troubleshooting. . . . . . . . . . . . . . . . . . . . . . 5
Quick Notes
Restrictions and Liabilities. . . . . . . . . . . . . . . . . . . . . . . . 6
• KAPA HiFi DNA Polymerase is extensively used
Note to Purchaser: Limited Product Warranty . . . . . . . . . 6 in next-generation sequencing (NGS) library
Note to Purchaser: Limited License . . . . . . . . . . . . . . . . . 6 amplification. If you are using this product in library
construction protocols, you may find KAPA HiFi
HotStart Library Amplification Kits more convenient.
These kits contain the KAPA HiFi HotStart DNA
Polymerase in a ReadyMix formulation, with or
without KAPA Library Amplification Primer Mix (10X)
for the amplification of Illumina® libraries. Please
refer to the KAPA HiFi HotStart Library Amplification
Kit Technical Data Sheet (KR0408) for details and a
standard library amplification protocol.
• KAPA HiFi PCR Kits contain the engineered
KAPA HiFi DNA Polymerase; developed for fast and
versatile high-fidelity PCR.
• The error rate of KAPA HiFi DNA Polymerase (as
determined by 454 sequencing) is 1 error per 3.6 x
106 nucleotides incorporated.
• Amplify targets up to 15 kb from genomic DNA or
20 kb from less complex targets.
• KAPA HiFi Buffers contain 2 mM MgCl2 at 1X.
• Use the KAPA HiFi Fidelity Buffer for routine high-
fidelity PCR, and the KAPA HiFi GC Buffer for GC-
rich and other difficult targets.
• Denature at 98°C for 20 sec per cycle.
• Optimal annealing temperatures are typically higher
than in other PCR buffer systems. Use an annealing
temperature gradient to determine the optimal
annealing temperature.
• To ensure the highest fidelity, use high quality DNA
and the lowest possible number of cycles.

Effective date: January 2019 For Research Use Only. Not for use in diagnostic procedures.
KAPA HiFi PCR Kit Technical Data Sheet
Product Description Product Applications
KAPA HiFi DNA Polymerase is a B-family DNA polymerase, The KAPA HiFi PCR Kit is ideally suited for:
engineered to have increased affinity for DNA, without the
• NGS library amplification
need for accessory proteins or DNA binding domains.
The intrinsic high processivity of the enzyme results in • Amplification of DNA fragments for Sanger sequencing
significant improvement in yield, speed and sensitivity (direct sequencing or sequencing of cloned PCR
when compared to wild-type B-family DNA polymerases. products)
In addition, the ability to amplify long fragments, as well as
• Amplification of DNA fragments to be cloned for protein
GC- and AT-rich targets, is significantly improved.
expression or genomic characterization
KAPA HiFi DNA Polymerase is supplied with two uniquely-
• Site-directed mutagenesis.
formulated PCR buffers for optimal performance. Both
buffers contain MgCl2 at a 1X concentration of 2  mM. For more information on these and other high-fidelity PCR
KAPA HiFi Fidelity Buffer is recommended for routine applications, please refer to KAPA HiFi Application Notes
high-fidelity PCR, whereas KAPA HiFi GC Buffer is available from www.sequencing.roche.com.
recommended for the amplification of GC-rich and other
difficult targets. Additives in the GC Buffer result in a two-
Product Specifications
fold decrease in fidelity when compared with the Fidelity
Buffer. Shipping and Storage
KAPA HiFi PCR Kits are designed for routine, high-fidelity KAPA HiFi PCR Kits are shipped on dry ice or ice packs,
PCR of a wide range of targets and fragment sizes. It depending on the country of destination. Upon arrival,
offers error rates approximately 100 times lower than store kit components at -15°C to -25°C in a constant-
wild-type Taq DNA polymerase, and higher success temperature freezer. When stored under these conditions
rates and yields than achievable with wild-type B-family and handled correctly, full activity of the kit is retained until
(proofreading) DNA polymerases. In addition, KAPA HiFi the expiry date indicated on the kit label.
requires significantly shorter cycling times than wild-type
Handling
B-family DNA polymerases.
KAPA HiFi buffers and enzymes contain isostabilizers and
KAPA HiFi DNA Polymerase has 5’g3’ polymerase and
may not freeze solidly, even when stored at -15°C to -25°C.
3’g5’ exonuclease (proofreading) activity, but no 5’g3’
This will not affect the shelf-life of the product. Nevertheless,
exonuclease activity. The strong 3’g5’ exonuclease
always ensure that the product has been fully thawed and
activity results in extremely high accuracy during DNA
mixed before use. Reagents may be stored at 2°C to 8°C
amplification. The error rate of KAPA HiFi DNA Polymerase
for short-term use (up to 1 month). Return to -15°C to
(determined by 454 sequencing) is 1  error per 3.6 x 106
-25°C for long-term storage. Provided that all components
nucleotides incorporated. This fidelity is approximately 100
have been handled carefully and not contaminated, the kit
times higher than that of wild-type Taq DNA polymerase,
is not expected to be compromised if left (unintentionally)
and up to ten times higher than that of other B-family DNA
at room temperature for a short period of time (up to
polymerases and polymerase blends.
3 days). Long-term storage at room temperature and 2°C
DNA fragments generated with KAPA HiFi DNA to 8°C is not recommended. Please note that reagents
Polymerase may be used for routine downstream analysis stored at temperatures above -15°C to -25°C are more
and applications, including restriction enzyme digestion, prone to degradation when contaminated during use, and
cloning and sequencing. PCR products generated with therefore storage at such temperatures is at the user’s
KAPA HiFi PCR Kits are blunt-ended, but may be 3’-dA- own risk.
tailed for cloning into TA-cloning vectors (see Important
Quality Control
Parameters: TA-cloning).
KAPA HiFi PCR Kits are subjected to stringent quality
control tests, are free of contaminating exo- and
endonuclease activity, and meet strict requirements with
respect to DNA contamination levels. Each batch of
KAPA HiFi DNA Polymerase is confirmed to contain <2%
contaminating protein (Agilent Protein 230 Assay).

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

KAPA HiFi PCR Kit Technical Data Sheet
Important Parameters Primer and Template DNA quality
Another critical factor for successful PCR with KAPA HiFi
Annealing temperature is primer design and quality. Primers should be carefully
Due to the high salt concentration in KAPA HiFi buffers, designed to eliminate the possibility of primer-dimer
the optimal annealing temperature for a given primer set is formation and nonspecific annealing as far as possible,
usually higher when compared to a different buffer system. and should have a GC content of 40 – 60%. Primers
When using the KAPA HiFi PCR Kit with a specific primer with GC content >60% may require higher denaturation
pair for the first time, determine the optimal annealing temperatures and/or longer denaturation times, while
temperature with annealing temperature gradient PCR. primers with GC content <40% may require annealing
We recommend a gradient from 60 – 72°C, although some temperatures <60°C, and/or increased MgCl2 and primer
assays may require even higher annealing temperatures. concentrations. Furthermore, primer sets should be
For assays with optimal annealing temperatures of 68°C or designed to have similar theoretical melting temperatures,
higher, two-step cycling may be performed at the optimal particularly for the 3 – 5 nucleotides at the 3'-terminal of
annealing temperature. Optimal annealing temperatures the primer.
below 60°C are rare, but may be required when using
primers with a high AT-content. NOTE: Always dilute and store primers in a buffered
solution (e.g. 10 mM Tris-HCl, pH 8.0 – 8.5) instead of
If a gradient PCR is not feasible, use an annealing PCR-grade water to limit degradation and maintain primer
temperature of 65°C as a first approach, and adjust the quality.
annealing temperature based on the results obtained:
High-quality template DNA is essential for high-fidelity
• If a low yield of only the specific product is obtained, amplification. Degraded, damaged, or sheared template
lower the annealing temperature in 1 – 2°C increments. DNA is particularly problematic when amplifying longer
• If nonspecific products are formed in addition to the fragments (>1 kb). To limit degradation and maintain
specific product, increase the annealing temperature template quality, always dilute and store DNA in a buffered
in 1 – 2°C increments. solution (e.g. 10 mM Tris-HCl, pH 8.0 – 8.5) instead of
PCR-grade water.
• If no product is formed (specific or nonspecific), reduce
the annealing temperature by 5°C. MgCl2 concentration Amplification from low-complexity templates, such as
may have to be increased. plasmid DNA, generally requires minimal optimization.
Applications based on low target copy numbers (e.g.
• If only nonspecific products are formed (in a ladder-like when amplifying single-copy genes from genomic
pattern), increase the annealing temperature by 5°C or templates, or when using cDNA as template) are generally
try recommendations for GC-rich PCR (see Important more challenging. For plasmid DNA, 1 – 10 ng template
Parameters: GC-rich PCR). per 25   µL reaction is sufficient, whereas up to 100 ng
complex genomic DNA or cDNA may be required.
NOTE: The optimal annealing temperature for a specific
amplicon is typically 5 – 6°C lower in the KAPA HiFi GC TA-cloning
Buffer than in the KAPA HiFi Fidelity Buffer. DNA fragments generated with the KAPA HiFi PCR Kit
may be used directly for blunt-end cloning, or cloning
MgCl2 concentration
using restriction endonucleases. For TA-cloning of KAPA
KAPA HiFi buffers contain a final (1X) MgCl2 concentration HiFi PCR products, first purify the PCR product to remove
of 2 mM, which is sufficient for most applications. the KAPA HiFi DNA Polymerase, as residual proofreading
Applications which are likely to require higher MgCl2 activity will remove any dA-overhangs added during the
concentrations include long PCR (>10 kb) and AT-rich A-tailing reaction. Perform A-tailing by combining the
PCR, as well as amplification using primers with a low GC purified PCR product, 1X Taq buffer (with 1.5 mM MgCl2),
content (<40%). 0.2 mM dATP and 1 U of Taq DNA polymerase and
GC-rich PCR incubating for 5 min at 72°C.
Use KAPA HiFi GC Buffer for the amplification of GC- NGS library amplification
rich targets. Alternatively, evaluate the KAPA HiFi Fidelity NGS library amplification differs from other high-fidelity
Buffer + 5% DMSO. Should neither of these result in PCR applications in three noteworthy ways: (i) unlike
successful amplification, perform reactions in both the genomic DNA or plasmids, templates are comprised of
KAPA HiFi Fidelity and GC Buffers, adding either 1X KAPA highly heterogenous populations of linear DNA or cDNA
Enhancer 1 (supplied with KAPA2G Robust PCR Kits) or fragments; (ii) the input copy number is orders of magnitude
1 M betaine to determine whether this improves yield and/ higher than in “conventional” PCR applications, and
or specificity. (iii) the aim is not to amplify a single amplicon with high
specificity, but to amplify a complex collection of library
fragments with minimal bias. For important parameters
relating to NGS library amplification with KAPA HiFi kits,
please refer to the KAPA HiFi HotStart Library Amplification
Kit Technical Data Sheet (KR0408).
For Research Use Only. Not for use in diagnostic procedures. 3
KAPA HiFi PCR Kit Technical Data Sheet
Standard PCR Protocol Step 3: Run the PCR
3.1 Perform PCR with the following cycling protocol:
IMPORTANT! The KAPA HiFi PCR Kit contains an
engineered B-family (proofreading) DNA polymerase and Step Temperature Duration Cycles
uniquely-formulated buffers, and requires specialized Initial denaturation 1
95ºC 3 min 1
reaction conditions. If these conditions are not adhered to,
reaction failure is likely. Refer to Important Parameters for Denaturation2 98ºC 20 sec
more information.
Annealing3,4 60 – 75ºC 15 sec 15 – 356
Step 1: Prepare the PCR master mix
1.1 KAPA HiFi reactions MUST be set up on ice since the Extension5 72ºC 15 – 60 sec/kb
high proofreading activity of the enzyme will result in Final extension 72ºC 1 min/kb 1
rapid primer degradation at room temperature.
1
Initial denaturation for 3 min at 95°C is sufficient for most applications. Use 5 min at
1.2 Ensure that all reagents are properly thawed and mixed. 95°C for GC-rich targets (>70% GC content).
2
KAPA HiFi buffers have a higher salt concentration than conventional PCR buffers,
1.3 Prepare a PCR master mix containing the appropriate which affects DNA melting. To ensure that complex and GC-rich targets are completely
volume of all reaction components common to all or a denatured, use a temperature of 98°C for denaturation during cycling.
subset of reactions to be performed. 3
In addition to DNA melting, the high-salt buffers also affect primer annealing. The
optimal annealing temperature for a specific primer set is likely to be different (higher)
1.4 Calculate the required volumes of each component than when used in a conventional PCR buffer. An annealing temperature gradient PCR
is recommended to determine the optimal annealing temperature with KAPA HiFi. If
based on the following table: gradient PCR is not feasible, anneal at 65°C as a first approach.
4
Two-step cycling protocols with a combined annealing/extension temperature in
Component 25 µL reaction1 Final conc. the range of 68 – 75°C and a combined annealing/extension time of 30 sec/kb may
be used.
PCR-grade water Up to 25 µL N/A 5
Use 15 sec extension per cycle for targets ≤1 kb, and 30 – 60  sec/kb for longer
fragments, or to improve yields.
5X KAPA HiFi Buffer
5.0 µL 1X 6
For highest fidelity, use ≤25 cycles. In cases where very low template concentrations
(Fidelity or GC)2 or low reaction efficiency results in low yields, 30 – 35 cycles may be performed to
produce sufficient product for downstream applications.
10 mM KAPA dNTP Mix 0.75 µL 0.3 mM each

10 μM Forward Primer 0.75 µL 0.3 μM

10 μM Reverse Primer 0.75 µL 0.3 μM

Template DNA3 As required As required

1 U/μL KAPA HiFi DNA

0.5 µL 0.5 U
Polymerase
1
Reaction volumes may be adjusted between 10 – 50 µL. For volumes other
than 25 μL, scale reagents down proportionally. Reaction volumes >50 µL are not
recommended.
2
KAPA HiFi Buffers contain 2 mM MgCl2 (1X). Additional MgCl2 may be added
separately. Use the GC Buffer only if the Fidelity Buffer gives poor results.
3
Use <100 ng genomic DNA (10 – 100 ng) and <1 ng less complex DNA (0.1 – 1 ng)
per 25 µL reaction as first approach.

Step 2: Set up individual reactions

2.1 Transfer the appropriate volumes of PCR master mix,
template and primer to individual PCR tubes or wells of
a PCR plate.
2.2 Cap or seal individual reactions, mix and centrifuge
briefly.

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

KAPA HiFi PCR Kit Technical Data Sheet
Appendix A - Troubleshooting

Symptoms Key parameters Solutions

No amplification or • Use the recommended 3 – 5 min initial denaturation at 95°C, and perform
low yield cycle denaturation for 20 sec at 98°C.
Cycling protocol
• Increase the extension time to a maximum of 1 min/kb.
• Increase the number of cycles.
Annealing temperature is too • Reduce the annealing temperature by 5°C, or try the GC Buffer.
high • Optimize the annealing temperature by gradient PCR.
• Excess template DNA chelates Mg2+. Either reduce the template
Template DNA quantity and concentration to <100 ng, or increase MgCl2.
quality • Check template DNA quality. Store and dilute in a buffered solution, not
water.
• Some primers anneal more efficiently than others. Increase the primer
Primer concentration concentration, or optimize MgCl2 to improve primer binding. Store and
dilute primers in a buffered solution, not water.
• Optimize MgCl2 concentration. AT-rich PCR typically requires more
MgCl2 concentration
MgCl2.
dNTP quality • dNTP quality is critical. Use only KAPA dNTPs supplied with the kit.
Nonspecific • Use <100 ng of DNA per reaction, or reduce the number of cycles.
amplification or Template DNA
smearing • Check template DNA quality.
• Excessive annealing and/or extension times will result in nonspecific
amplification, typically of bands larger than the target band. Reduce the
Cycling protocol annealing and extension times to a minimum of 10 sec each.
• Reduce the number of cycles.
• A sub-optimal annealing temperature will result in nonspecific amplicons
Annealing temperature is too
that are typically smaller than the target band. See Important Parameters:
low
Annealing Temperature.
• Use the GC Buffer, or add 5% DMSO to Fidelity Buffer.
Target GC content • Add 1X KAPA Enhancer 1 or 1 M betaine to reactions with Fidelity and/or
GC Buffer to facilitate melting of GC-rich templates.
• Do not exceed 0.5 U of KAPA HiFi DNA Polymerase per 25 µL reaction.
Enzyme concentration
This results in smearing and nonspecific amplification.
• Some primers anneal more efficiently than others. Decrease the primer
Primer concentration
concentration. Store and dilute primers in a buffered solution, not water.

For Research Use Only. Not for use in diagnostic procedures. 5

KAPA HiFi PCR Kit Technical Data Sheet
Restrictions and Liabilities Note to Purchaser: Limited Product Warranty
This technical data sheet is provided “as is” and Any product that does not meet the performance standards
Kapa Biosystems assumes no responsibility for stated in the product specification sheet will be replaced at
any typographical, technical, or other inaccuracies. no charge. This warranty limits our liability to the replacement
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To the maximum extent permitted by applicable law, Kapa
provided by Kapa Biosystems. Kapa Biosystems shall have
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information contained herein, including but not limited to
inability to use any product.
the implied warranties of merchantability and fitness for
a particular purpose. Kapa Biosystems shall not be liable
for errors or for incidental or consequential damages in Note to Purchaser: Limited License
connection with the furnishing, use, or performance of this KAPA HiFi PCR Kits are developed, designed and sold
document or of any information contained herein. exclusively for research purposes. Neither the product,
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6 For Research Use Only. Not for use in diagnostic procedures.