Consolidate PCR protocols and increase success rates with KAPA2G Robust HotStart DNA Polymerase and ReadyMix. Download the new application note for Single Protocol PCR. Download here>>

Consistently amplify your most difficult GC-rich amplicons with KAPA2G Robust DNA Polymerase. Download the new application note for Routine GC-rich PCR. Download here>>

Revolutionize your Colony PCR work flow! Download the new application note for E. coli and yeast Colony PCR using KAPA2G Robust DNA Polymerase. Download here>>

Product Description

The KAPA2G Robust DNA Polymerase is a highly robust and versatile second-generation enzyme derived through a process of molecular 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 GC- and AT-rich templates and amplicons, as well as improved tolerance to common PCR inhibitors.

In the HotStart formulation, the enzyme is 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.

KAPA2G Robust Kits supplied with KAPA2G Buffer A and KAPA2G Buffer B and the proprietary additive, KAPA Enhancer 1, offer extended optimization options for diverse and difficult templates. Kits also contain KAPA2G GC Buffer, a novel buffer formulated specifically for GC-rich templates and amplicons. Like wild-type Taq, KAPA2G Robust DNA Polymerase has 5’-3’ polymerase and exonuclease activities, but no 3’-5’ exonuclease (proofreading activity). The fidelity of KAPA2G Robust DNA Polymerase is similar to that of wild-type Taq.

Product Applications

KAPA2G Robust DNA Polymerase Kits, and HotStart formulations thereof, are recommended for all standard end-point PCR assays, particularly those in which wild-type Taq DNA polymerase does not perform satisfactorily. Kits are particularly suited for:

• Amplification from templates with a high GC- or AT content.
• Consolidation of PCR protocols and reaction conditions while improving success rates.
• Templates containing common PCR inhibitors (e.g. salts, urea, SDS and ethanol).
• Amplification from crude samples, e.g. buccal swabs, cultured mammalian, yeast or bacterial cells (Colony PCR).
• Optimization of low yield or low specificity assays.

Amplicons generated with KAPA2G Robust DNA Polymerase are suitable for routine downstream applications, including restriction enzyme digestion, cloning and sequencing.

Product Performance

Consolidate PCR protocols and increase success rates with a single enzyme

The improved processivity and tolerance to common PCR inhibitors of the KAPA2G Robust DNA Polymerase offers consistent amplification, high yields and wide coverage of both easy and challenging amplicons. The unique features of the enzyme supports versatile and robust amplification of a broad range of AT- and GC-rich targets and allows for the simplification of PCR workflows, through the consolidation of reagents and protocols, while increasing success rates and turnaround time.

A total of 96 amplicons were amplified from human genomic DNA, using the recommended reaction setup and cycling protocol for KAPA2G Robust HotStart ReadyMix, or the standard reaction conditions for wild-type Taq. The overall success rate achieved with KAPA2G Robust HotStart ReadyMix (in a total cycling time of 36 min) was 96%, compared to 66% achieved in >1 hour cycling time with wild-type Taq. The data clearly illustrates the expanded amplification range of KAPA2G Robust: high success rates were achieved across the full spectrum of GC contents, whereas wild-type Taq showed poor results with AT-rich amplicons and amplicons with a GC content >60%. Numbers in brackets indicate the number of primer sets representing each subset of amplicons (by GC content) in the experiment.

Half of each of the PCR products obtained with 72 of the 96 primer sets used in this study were electrophoresed in a 1% TBE-agarose gel. Amplicons were loaded in order of increasing GC content, with the lowest GC content (27%, blue) at the top left hand side and the highest GC content (84%, red) at the bottom right hand side of each composite gel image. Primers selected for this study had variable primer lengths, sequence composition, theoretical melting temperatures and other design features. Some primers contained 5'-tails for post-PCR sequencing using M13 or other standard sequencing primers. KAPA2G Robust HotStart ReadyMix reactions (25 µl) were performed as outlined in Tables 1 and 2. Wild-type Taq reactions (25 µl, containing 0.5 U Taq per reaction) were performed in Taq reaction buffer (1.5 mM MgCl 2 at 1X), using the same final primer and dNTP concentrations as for KAPA2G Robust. All reactions contained 25 ng human genomic DNA. 5% DMSO was included in all reactions (KAPA2G Robust and Taq) targeting amplicons with a GC content >70%. 

Greatly improved tolerance to common PCR inhibitors

KAPA2G Robust DNA Polymerase was engineered for high performance in chemically complex reaction conditions. The result is superior tolerance to a wide range of common PCR inhibitors, when compared to wild-type Taq polymerase and so-called “robust” polymerase blends. In addition to the examples below, KAPA2G Robust HotStart DNA Polymerase shows improved tolerance to other inhibitors, including KCl, sodium acetate, isopropanol and phenol (at high template and enzyme concentrations).

Amplification of a 1.5 kb fragment from 1 pg plasmid DNA in the presence of four common PCR inhibitors using the KAPA2G Robust HotStart PCR kit (top), wild-type hot start Taq polymerase (middle) or a “robust” blend of thermostable DNA polymerases (bottom). All reactions contained 0.5 units of enzyme per 25 µl reaction, except for reactions with the polymerase blend (0.625 units per reaction). KAPA2G Robust HotStart Buffer B was used throughout, with the addition of KAPAEnhancer 1 for reactions containing SDS. Cycling was performed with an Eppendorf Mastercycler epgradient S, using a standard 3-step cycling profile (35 cycles) with an annealing temperature of 64ºC and 1.5 min extension time per cycle for all enzymes.

Unrivalled performance in Colony PCR

The improved inhibitor tolerance of KAPA2G Robust translates into unrivalled performance in Colony PCR, a PCR application that is prone to failure and inconsistency due to the presence of inhibitors. With the KAPA2G Robust PCR kit, a 100% success rate in Colony PCR is achievable, starting from plated bacterial colonies or overnight cultures.

Amplification of a 2.7 kb gene from recombinant E. coli colonies using the KAPA2G Robust HotStart PCR Kit (top), wild-type hot start Taq (middle) or a blend of thermostable DNA polymerases (bottom). Three single colonies from each of four commonly used E. coli strains (BL21, DH5a, DH10B and JM109) were picked from LB-agar + ampicillin plates and resuspended in PCR grade water. 1 µl resuspended bacterial cells was used as template in a 25 µl PCR. All reactions contained 0.5 units of enzyme, except for reactions with the polymerase blend (0.625 units per reaction). KAPA2G Robust HotStart Buffer A was used in all KAPA2G Robust HotStart reactions. Cycling was performed with a G-Storm GS1 thermocycler with fast block, using a standard 3-step cycling profile (35 cycles) with 1 min/kb extension time for all enzymes.

KAPA2G Robust DNA Polymerase PCR kits offer higher yields and sensitivity

The higher processivity and specific activity of KAPA2G Robust DNA Polymerase results in higher yields per unit of enzyme, which often translates into improved sensitivity. Higher yields and sensitivity are also achieved through the use of novel buffers, which have been formulated for optimal annealing specificity. For certain assays, specificity and efficiency may be improved further through the use of KAPAEnhancer 1 in combination with KAPA2G Buffer A or KAPA2G Buffer B.

Amplification of a 5 kb lambda (left) and a 2.7 kb human (right) amplicon using the KAPA2G Robust HotStart PCR Kit or wild-type hotstart Taq polymerase. A10-fold dilution series of template DNA (106 to 103 copies lambda DNA or 30,000 to 30 copies human DNA, respectively) was included in each experiment. All reactions contained 0.5 units of enzyme per 25 µl reaction, except for 5 kb reactions with KAPA2G Robust HotStart, in which only 0.25 units of enzyme were used. For the 2.7 kb assay, KAPAEnhancer 1 was included in KAPA2G Robust HotStart reactions. Cycling was performed with a G-Storm GS1 thermocycler with a fast block (5 kb amplicon) or Eppendorf Mastercycler epgradient S (2.7 kb amplicon), using standard 3-step cycling profiles (35 cycles) with extension rates of 1 - 2 min/kb.

Frequently Asked Questions

1. What are the most common causes of poor results (non-specific amplification, smearing or reaction failure) with KAPA2G Robust?

• Template DNA quality is too low. High yields of target amplicons should not be expected when using degraded DNA, DNA with a low concentration and/or DNA containing high levels of PCR inhibitors.
• Suboptimal 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.
• Suboptimal 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.
• Suboptimal annealing time. Use 15 sec per cycle for good quality DNA. This may be reduced to 10 sec per cycle for slow cyclers or reduced reaction volumes (<25 µl). For difficult samples, annealing times may be increased to 30 sec per cycle.
• Too much starting template. For high quality DNA, 1 – 50 ng genomic DNA or ≤1 ng plasmid/lambda DNA per 25 µl reaction should be sufficient 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.
• Suboptimal amount of enzyme. 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 concentrations are not optimal. Use a final concentration of 0.2 mM of each dNTP and 0.5 µM of each primer.
• Target is too long. 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 KAPAHiFi DNA Polymerase.

2. What are the recommended applications for KAPA2G Robust PCR Kits?
KAPA2G Robust DNA Polymerase is recommended for all standard end-point PCR assays. The robust performance of the enzyme allows for consolidating PCR protocols and reaction conditions across a wide variety of primer and amplicon types, while increasing success rates. It is particularly suited for the following applications:

• Amplicons with a high GC- or AT content.
• 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.
• Improving yield and/or sensitivity in assays where template quality or primer design is problematic.

3. Why is the KAPA2G Robust DNA Polymerase the preferred enzyme for Robust PCR?
KAPA2G Robust DNA Polymerase is a second-generation enzyme that was engineered through a process of molecular 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 shows greatly 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. Three high-yield buffers and one proprietary PCR additive supplied in the kit offer higher yields and sensitivity across a wide variety of PCR assays, as well as extended optimization options for difficult templates or amplicon types.

4. Does it matter whether I use KAPA2G Robust or KAPA2G Robust HotStart?
KAPA2G Robust HotStart is an antibody-mediated hot start formulation of KAPA2G Robust DNA Polymerase. Both enzymes are supplied with the same three reaction buffers and a proprietary additive, KAPAEnhancer 1, for a variety of optimization options. The non-HotStart and HotStart formulations should give similar results in most assays. However, for workflows that require room temperature setup, the HotStart formulation must be used.

5. Which KAPA2G Buffer should I use?

• KAPA2G Robust enzymes are supplied with three distinct reaction buffers, and the proprietary additive, KAPAEnhancer 1. This offers five buffer/additive combinations for optimization:
• KAPA2G Buffer A
• KAPA2G Buffer A + KAPAEnhancer 1
• KAPA2G Buffer B
• KAPA2G Buffer B + KAPAEnhancer 1
• KAPA2G GC Buffer (do not combine with KAPAEnhancer 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 maybe used as a starting point:

• Use KAPA2G Buffer A (with or without KAPAEnhancer 1) for good quality DNA, amplicons with a GC content <65%.
• Use KAPA2G Buffer B (with or without KAPAEnhancer 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 recalcitrant to amplification, the following additional buffer/additive combinations may be tried:

• KAPA2G GC Buffer + 4% DMSO
• KAPA2G Buffer A + KAPAEnhancer 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.

6. What should I do if PCR products generated with KAPA2G Robust contain a high background of non-specific amplicons or high molecular weight smears?
The high specific activity and processivity of KAPA2G Robust DNA Polymerase may sometimes result in a higher background of small or large non-specific amplicons or high-molecular weight smears when compared to products obtained with wild-type Taq (or hot start formulations thereof) under similar conditions. Since KAPA2G Robust is a second-generation enzyme, some optimization may be required when it is used in assays originally designed for wild-type Taq. To improve the yield of the specific amplicon, one or more of the following may be done:

• Reduce the annealing time to 10 – 15 sec 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, 1 – 50 ng genomic DNA or ≤1 ng plasmid/lambda DNA per 25 µl reaction should be sufficient for most applications.
• Reduce the number of cycles.
• Reduce the amount of enzyme per reaction.
• Try one of the other KAPA2G Robust buffers 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.

7. Should I always use 0.5 units of KAPA2G Robust DNA Polymerase per 25 μl reaction?
0.5 units of KAPA2G Robust DNA Polymerase per 25 μl reaction (or proportionally more or less for larger or smaller reaction volumes) should be sufficient for most standard PCR applications. However, in some cases more or less enzyme may yield better results. To improve yields and sensitivity, increase the amount of enzyme 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).
• Results may be improved by reducing 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.

8. What is the recommended extension time for KAPA2G Robust DNA Polymerase?
An extension time of 15 sec/kb per cycle should be 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.

9. Do I have to include MgCl2 in my reaction setup?
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.

10. Can I store KAPA2G Robust PCR Kits at room temperature or 4 °C?
The recommended temperature for long-term storage of KAPA2G Robust enzymes, KAPA2G Buffers A, B and GC Buffer, KAPAEnhancer 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 a month). Don’t worry if you’ve left any component of the kit on your bench overnight or over the weekend – it will still work fine (but don’t make a habit of it!)

11. 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.

12. When and how should I use KAPA Enhancer 1 in my assay?
KAPA Enhancer 1 is a proprietary PCR additive (DNA destabilizer) that improves reaction efficiency and specificity for some, but not all, primer-template combinations. It is supplied as a 5X solution and should always be used at a final concentration of 1X. 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.

13. Can I use other PCR additives with KAPA2G Robust?
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 addi tives 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.
• KAPAEnhancer 1 has similar properties to betaine and may be used at a 1X concentration instead of betaine in any assay shown to benefit from the inclusion of betaine. Do not combine KAPAEnhancer 1 and betaine.
• The inclusion of 5% glycerol or 0.1% Tween 20 in reactions often improves the amplification of longer fragments. However, KAPAHiFi is recommended for the robust amplification of amplicons >3 kb.

14. What is the best strategy for GC-rich PCR?

• Use 1 unit enzyme per 25 μl reaction or equivalent.
• First try 1X KAPA2G GC Buffer without any additives.
• For particularly recalcitrant 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 min at 95 °C or pre-denature the template. Denature for 15 - 30 sec at 95°C per cycle.
• Please refer to the Application Note: Routine GC-Rich PCR for more information.

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

• Use KAPA2G Buffer B or KAPA2G GC Buffer for GC-rich amplicons. KAPA Enhancer 1 may improve results in some cases.
• Use 0.5 – 1.0 U enzyme per 25 μl reaction or equivalent.
• 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.
• 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 min (95 °C) and 10 – 15 sec denaturation time (95 °C) per cycle.
• Use an annealing time of 10 – 15 sec per cycle.
• Use an extension time (72 °C) of 5 sec 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.

16. What is the best strategy for yeast Colony PCR?

• Lyse yeast cells (overnight colonies or cultures) in 0.1 M NaOH or Zymolase for at least 5 min at 37 °C.
• Use KAPA2G Buffer B or KAPA2G GC Buffer for GC-rich amplicons. KAPA Enhancer 1 may improve results in some cases.
• Use 1.0 U enzyme per 25 μl reaction or equivalent.
• 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.
• Include 2.5 μl of the lysed yeast cell suspension per 25 μl reaction.
• Use an initial denaturation time of 5 min (95 °C) and 30 sec denaturation time (95 °C) per cycle.
• Use an annealing time of 10 – 15 sec per cycle.
• Use an extension time (72 °C) of 15 sec 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.
• Please refer to the Application Note: Colony PCR for more information.

17. 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. Vortex for 15 seconds.

• Incubate for 10 minutes at 95 °C. After incubation, vortex for 15 seconds.
• Centrifuge for 1 min 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 (5 dilutions plus the undiluted sample) in PCRs with KAPA2G Robust or KAPA2G Robust HotStart, using the reaction setup conditions in the Technical Data Sheet.
• Start with the following cycling profile: initial denaturation: 3 min at 95 °C, denaturation: 15 sec (95 °C) per cycle; annealing: 15 sec (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 KAPAEnhancer 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 a 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.

18. 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 in this regard. Multiplex 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 parameters (e.g. buffer, MgCl2 concentration, relative concentration of each primer pair, enzyme and template concentrations, annealing temperature, extension time and number of cycles) is likely to be required.

19. How can I go about optimizing 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 and 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 used 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 amount 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.

20. Can PCR products generated with the KAPA2G Robust be digested or 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. For best results, purification of PCR products using any standard PCR cleanup kit is recommended prior to RE digestion or sequencing.

21. Can PCR products generated with the KAPA2G Robust be cloned?
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 prior to cloning.
The error rate (fidelity) of the KAPA2G Robust DNA Polymerase is the same as that of wild-type Taq polymerase. The KAPA2G Robust PCR Kit may therefore be used for the amplification of short fragments (<1 kb) for cloning. To limit the introduction of mutations due to the inherent error rate of Type A polymerases, start with the highest possible target copy number and limit the number of cycles to 25 or less. If the fidelity of the cloned product is not important, any sized fragment amplified with KAPA2G Robust DNA Polymerase may be cloned.
KAPA HiFi DNA Polymerase (an engineered B-family polymerase with proofreading activity) is recommended for the amplification of longer DNA for cloning where fidelity is important.

22. Can PCR products generated with KAPA2G Robust PCR Kits be analysed by dHPLC?
Yes. PCR products generated with KAPA2G Robust or KAPA2G Robust HotStart, using KAPA2G Buffer A (with or without KAPAEnhancer 1), KAPA2G Buffer B (with or without KAPAEnhancer 1) of 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.