Technical Data Sheet

KAPA2G Fast ReadyMix PCR Kit Kit Codes and Components

KR0374_S – v2.17 KK5101

(1.25 mL)
Product Description KK5102 KAPA2G Fast ReadyMix with dye (2X)
(6.25 mL) (contains 1.5 mM MgCl2 at 1X)
KAPA2G Fast DNA Polymerase is a second-generation
enzyme engineered through a process of directed KK5103
(25 mL)
evolution. KAPA2G Fast was engineered for higher
processivity and speed, offering significantly faster
extension rates than wild-type Taq DNA polymerase. The
enzyme is supplied with a buffer specifically formulated for Quick Notes
the unique characteristics of the enzyme. This optimized • KAPA2G Fast ReadyMix PCR Kits contain the
buffer offers improved yields, specificity, and sensitivity engineered KAPA2G Fast DNA Polymerase,
compared to typical wild-type Taq buffers. developed for fast PCR.
In the ReadyMix PCR Kit, KAPA2G Fast DNA Polymerase • Use 1 sec extension time for amplicons <1 kb
is supplied in a convenient 2X ReadyMix format, and 15 sec/kb for longer amplicons, and save
containing all reaction components except primers and 20–70% of total reaction time.
template. The ReadyMix contains KAPA2G Fast DNA • No need for specialized instrumentation or PCR
Polymerase (0.5 U per 25 µL reaction) in a proprietary consumables.
reaction buffer containing dNTPs (0.2 mM of each dNTP at • Optimized buffer system offers improved yields,
1X), MgCl2 (1.5 mM at 1X), and stabilizers. The ReadyMix specificity and sensitivity, facilitating efficient primer
also contains two inert tracking dyes to enable direct annealing across a wide range of primer lengths,
loading of PCR products onto agarose gels for analysis by GC contents, and melting temperatures.
electrophoresis, without the need to add a DNA loading
• For high reaction efficiency, do not exceed 25 μL
solution.
reaction volumes.
KAPA2G Fast ReadyMix PCR Kits are designed for fast • KAPA2G Fast ReadyMix contains 1.5 mM MgCl2 at
PCR, in which total reaction times are 20–70% shorter 1X.
than those of conventional PCR assays performed with
• KAPA2G Fast ReadyMix includes two inert tracking
wild-type Taq DNA polymerase. This can be achieved
dyes to allow direct loading onto agarose gels.
without sacrificing reaction performance, and does not
require specialized PCR consumables or thermocyclers. • Reaction products are 3’-dA-tailed and may be
cloned into TA cloning vectors.
DNA fragments generated with KAPA2G Fast DNA
Polymerase have the same characteristics as DNA Product Applications
fragments generated with wild-type Taq DNA polymerase,
and may be used for routine downstream analyses or Most existing PCR assays performed efficiently with wild-
applications, including restriction enzyme digestion, type Taq DNA polymerase may be converted to fast PCR
cloning and sequencing. Like wild-type Taq, KAPA2G Fast assays with the KAPA2G Fast ReadyMix by following the
has 5’g3’ polymerase and 5’g3’ exonuclease activities, protocol provided in this document.
but no 3’g5’ exonuclease (proofreading) activity. The The following assays are likely to be unsuitable for fast
fidelity of KAPA2G Fast is similar to that of wild-type Taq; PCR with the KAPA2G Fast ReadyMix PCR Kit:
it has an error rate of approximately 1 error per 1.7 x 105
nucleotides incorporated. PCR products generated with • Amplification of long fragments (>1 kb) from low target
KAPA2G Fast are 3’-dA-tailed and may be cloned into TA copy numbers
cloning vectors. • Amplification of highly GC-rich fragments (>70%)
Other available KAPA2G Fast products include: • Amplification with primers that are prone to nonspecific
• KAPA2G Fast PCR Kits annealing

• KAPA2G Fast HotStart PCR Kits • Multiplex PCR

• KAPA2G Fast HotStart ReadyMix • Amplification from template samples that contain PCR
inhibitors.
• KAPA2G Fast Multiplex Mix
• KAPA2G Fast Genotyping Mix. Although conversion to fast PCR is possible for these
assay types, and other assays that do not work well with
Visit www.sigmaaldrich.com for additional product wild-type Taq, it is not recommended since significant
information, or contact Technical Support at optimization may be required.
sigma-aldrich.com/techservice for advice on the best
KAPA2G Fast product for your application.
Effective date: September 2017 For Research Use Only. Not for use in diagnostic procedures.
KAPA2G Fast ReadyMix PCR Kit Technical Data Sheet
Standard PCR Protocol Step 3: Run the PCR
IMPORTANT! The KAPA2G Fast ReadyMix contains an • Perform PCR with the following cycling protocol:
engineered DNA polymerase and uniquely-formulated
buffer, and requires specialized reaction conditions. If Step Temperature Duration Cycles
these conditions are not adhered to, reaction failure is
Initial denaturation 1
95ºC 3 min 1
likely. Refer to Important Parameters for more information.
Denaturation 95ºC 10–15 sec
Step 1: Prepare the PCR master mix
Annealing2 60ºC 10–15 sec 25–404
• Due to the high activity of the KAPA2G Fast DNA
Polymerase at room temperature, reactions should be Extension3 72ºC 1–15 sec/kb
set up on ice to limit nonspecific amplification during
setup. Final extension 72ºC 1 min/kb 1

• Ensure that all reagents are properly thawed and mixed. 1

Initial denaturation for 3 min at 95°C is sufficient for most applications. Use 5 min
at 95°C for GC-rich targets (>70% GC content).
• Prepare a PCR master mix containing the appropriate 2
KAPA2G Fast ReadyMix is uniquely formulated to facilitate primer annealing
volume of all reaction components common to all or a across a wide range of primer and amplicon lengths and GC contents. Use 60°C as
subset of reactions to be performed. a first approach, and adjust only if necessary.

• Calculate the required volumes of each component 3

The denaturation, annealing, and extension times are dependent on the
based on the following table: thermocycler ramp speed, and the yield required. Refer to the table in Important
Parameters: Cycling protocol for guidelines on the recommended extension time.

Component 25 µL reaction1 Final conc. 4

The number of cycles required is dependent on the size of the amplicon, and the
amount of template copies per reaction. A 35-cycle PCR can typically amplify a
high yield of product from 100 copies of template.
PCR-grade water Up to 25 µL N/A

2X KAPA2G Fast
ReadyMix2
12.5 µL 1X Product Specifications
10 μM Forward Primer 1.25 µL 0.5 μM Shipping, storage and handling
KAPA2G Fast ReadyMix PCR Kits are shipped on dry ice
10 μM Reverse Primer 1.25 µL 0.5 μM or ice packs, depending on the country of destination.
Upon arrival, store kit components at --15°C to -25°C in
Template DNA3 As required As required a constant-temperature freezer. When stored under these
conditions and handled correctly, full activity of the kit is
1
For volumes smaller than 25 μL, scale reagents down proportionally. Reaction
volumes >25 µL are not recommended. retained until the expiry date indicated on the kit label. The
KAPA2G Fast ReadyMix contains isostabilizers and may
2
KAPA2G Fast ReadyMix contains 1.5 mM MgCl2 at 1X. Reactions may be not freeze solidly, even when stored at -15°C to -25°C.
supplemented with additional MgCl2 if required.
This will not affect the shelf-life of the product.
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.
Always ensure that the product has been fully thawed and
mixed before use. Reagents may be stored at 2°C to 8°C
for short-term use (up to 1 month). Return to -15°C to
Step 2: Set up individual reactions -25°C for long-term storage. Provided that the ReadyMix
has been handled carefully and not contaminated, the kit
• Transfer the appropriate volumes of PCR master mix,
is not expected to be compromised if left (unintentionally)
template and primer to individual PCR tubes or wells
at room temperature for a short period of time (up to
of a PCR plate.
3 days). Long-term storage at room temperature and 2°C
• Cap or seal individual reactions, mix and centrifuge to 8°C is not recommended. Please note that reagents
briefly. stored at temperatures above -15°C to -25°C are more
prone to degradation when contaminated during use, and
therefore storage at such temperatures is at the user’s
own risk.
Quality control
Each batch of KAPA2G Fast DNA Polymerase is confirmed
to contain <2% contaminating protein (Agilent Protein 230
Assay). KAPA2G Fast ReadyMix is subjected to stringent
quality control tests, is free of contaminating exo- and
endonuclease activity, and meets strict requirements with
respect to DNA contamination levels.

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

KAPA2G Fast ReadyMix PCR Kit Technical Data Sheet
Important Parameters Annealing temperature
Cycling protocol KAPA2G Buffer A is designed to facilitate primer annealing
across a wide range of primer and amplicon lengths,
KAPA2G Fast ReadyMix is capable of amplifying fragments and GC contents. This means that for most assays, an
of up to 1 kb in size with a 1 sec/cycle extension time. annealing temperature of 60°C can be used with high
Recommendations for hold times are based on the ramp success rates. However, should 60°C not produce the
rate of the thermocycler, as well as the yield required. desired result, annealing temperatures may be optimized
Thermocyclers with ramp speeds higher than 3°C/sec are with gradient PCR, or adjusted as follows:
considered fast, while slow cyclers typically have ramp
rates from 1–3°C/sec. The slower the ramp speed, the • If a low yield of only the specific product is obtained,
shorter the hold time required. lower the annealing temperature in 2°C increments.

Standard cycling Maximum speed • If nonspecific products are formed in addition to the
(fast cyclers) (slow cyclers) Maximum yield (all specific product, increase the annealing temperature
cyclers) in 2°C increments.
Ramp rate >3°C/sec Ramp rate <3°C/sec
Denature 15 sec Denature 10 sec Denature 15 sec • If no product is formed (specific or nonspecific), reduce
Anneal 15 sec Anneal 10 sec Anneal 15 sec the annealing temperature by 5°C. MgCl2 concentration
may have to be increased.
Extend 5 sec Extend 1 sec Extend 15 sec
• If only nonspecific products are formed (in a ladder-like
Using excessive extension times may result in nonspecific pattern), increase the annealing temperature by 5°C or
amplification, smearing, primer dimer formation, and over- try recommendations for GC-rich PCR (see Important
amplification. If low yields are obtained even with a 15 sec Parameters: GC-rich PCR).
extension time, increase to a maximum of 30 sec/cycle, in
5 sec increments. For amplicons >1 kb in size, start with Primers and template DNA
15 sec/kb, and increase to a maximum of 1 min/kb, in
15 sec increments. Primers should be designed to eliminate the possibility
of primer-dimer formation and nonspecific annealing,
In addition to extension time, the annealing time is critical and should have a GC content of 40–60%. Primers with
to ensure success. At temperatures typically used for a GC content >60% may require higher denaturation
annealing (~60°C), KAPA2G Fast DNA Polymerase has temperatures and/or longer denaturation times, while
much higher activity than wild-type Taq DNA polymerase. primers with a GC content <40% may require annealing
Therefore, the use of excessive annealing times often temperatures <60°C, and/or increased MgCl2 and primer
results in the same effects as excessive extension times. concentrations. Furthermore, primer sets should be
Typically, the formation of nonspecific products that are designed to have similar theoretical melting temperatures.
larger than the target band indicates that the annealing
time used is too long. High-quality template DNA is essential for fast PCR.
Degraded, damaged, or sheared template DNA is
The number of cycles to use is dependent on the number particularly problematic when amplifying longer fragments
of template copies present at the beginning of the reaction. (>1 kb).
For routine applications, 35 cycles is sufficient for a high
yield of product. However, if the template DNA contains NOTE: Always dilute and store primers and template DNA
a high number of copies, cycle numbers may be reduced in a buffered solution (e.g. 10 mM Tris-HCl, pH 8.0–8.5)
accordingly. instead of PCR-grade water to limit degradation and
maintain quality.
MgCl2 concentration Amplification from low-complexity templates, such as
KAPA2G Buffer A contains 1.5 mM MgCl2 at 1X, which plasmid DNA, generally requires minimal optimization.
is sufficient for most applications. Applications which Applications based on low target copy numbers (e.g.
are likely to require higher MgCl2 concentrations include when amplifying single-copy genes from genomic
longer amplicons (>2 kb) and AT-rich PCR, as well as templates, or when using cDNA as template) are generally
amplification using primers with a low GC content (<40%). more challenging. For plasmid DNA, 1–10 ng template per
25  µL reaction is sufficient, whereas up to 100 ng complex
Amplicon size
genomic DNA or cDNA may be required.
For highly efficient fast PCR, we recommend using
amplicons that are <1 kb in size, with GC content in the GC-rich PCR
range of 35–65%. Longer assays can be converted to fast For GC-rich amplicons, supplement reactions with 5%
PCR, but may require significant assay optimization. DMSO. Should this not result in successful amplification,
the KAPA2G Robust PCR Kit, which is optimized for GC-
rich PCR, may be used.

For Research Use Only. Not for use in diagnostic procedures. 3

KAPA2G Fast ReadyMix PCR Kit Technical Data Sheet

Troubleshooting

Symptoms Possible causes Solutions

No amplification or Increase the extension time to a maximum of 30 sec per cycle (in 5  sec
low yield increments) for amplicons <1 kb in size. For larger amplicons, increase to a
Cycling protocol maximum of 60 sec/kb (in 15 sec increments).
Increase the number of cycles.

Annealing temperature is too Reduce the annealing temperature by 5°C.

high Optimize the annealing temperature by gradient PCR.

Excess template DNA chelates Mg2+. Either reduce the template concentration
Template DNA quantity and 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.

MgCl2 Optimize MgCl2 concentration. AT-rich PCR typically requires more MgCl2.

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.

Target GC content Supplement reactions with 5% DMSO, or try the KAPA2G Robust PCR Kit.

Some primers anneal more efficiently than others. Decrease the primer
Primer concentration concentration.
Store and dilute primers in a buffered solution, not water.

Manufacturing, R&D Technical Support

Cape Town, South Africa sigma-aldrich.com/techservice
Tel: +27.21.448.8200
Fax: +27.21.448.6503

© 2017 KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners.

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