KAPA2G Robust PCR Kits

Streamline your PCR workflows by consolidating protocols and reaction conditions, increase pass rates, amplify difficult templates without bias, and increase sensitivity – all with a single enzyme.

The second-generation KAPA2G Robust DNA Polymerase was evolved to solve - achieve consistent amplification across a broad range of amplicon types (both GC and AT-rich). The versatility and robustness of the polymerase allows for consolidation of PCR cycling protocols and reaction conditions while increasing success rates. The high performance of the KAPA2G Robust DNA Polymerase eliminates the need for multiple enzymes and protocols - standardize your PCRs with a single enzyme solution.

The KAPA2G Robust DNA Polymerase offers:

  • Robust performance across a wide range of GC- and AT-rich templates
  • Increased PCR success rates
  • Consolidated PCR protocols and reaction conditions with a single enzyme
  • Improved tolerance to common PCR inhibitors for crude samples and/or DNA extractions
  • Consistent Colony PCR from E. coli and yeast
  • Higher yield and sensitivity per unit of enzyme
  • Three novel buffers and a proprietary PCR enhancer offer extended optimization options for the most difficult templates

KAPA2G Robust DNA Polymerase is also available with HotStart technology for improved specificity during prolonged benchtop setup or liquid handling.

I work in a yeast genetics lab at Harvard Medical School where we had largely abandoned the use of colony PCR after finding it too unreliable - success was just too variable to consider it a useful technique.  This changed after trying a sample of KAPA2G Robust HotStart DNA Polymerase.  We found the success rate with this enzyme to be very high, even with difficult templates.  Now, 2G Robust has become our primary enzyme, replacing a whole catalog of other polymerases that we used to keep in stock.  We have again begun to use colony PCR regularly, and the only enzyme we use for it is KAPA2G Robust HotStart.

- Dan Spatt, Research Manager, Winston Lab, Department of Genetics, Harvard Medical School, Boston MA

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.