KAPA2G Fast PCR Kits

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A second-generation DNA polymerase engineered for the ultimate in speed and performance.

KAPA2G Fast DNA Polymerase is a second-generation enzyme derived through a process of molecular evolution. The KAPA2G Fast DNA Polymerase was engineered for higher processivity and speed, offering significantly faster extension rates and improved PCR success rates than wild-type Taq polymerase. KAPA2G Fast is well suited for both routine and high-throughput PCR, achieving higher yields and improved sensitivity than competitor enzymes across a wide range of amplicon types.

KAPA2G Fast DNA Polymerase and optimized buffer system offers:

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

The use of this second-generation polymerase allowed us to increase our throughput by 50%, while maintaining and even improving the quality of our results. This is really important because it accelerates the process of mutation detection.

- Dr. Adriana Heguy, Manager of the Geoffrey Beene Translational Oncology Core Facility at Memorial Sloan Kettering Cancer Center

Code Description Kit Contents Qty Unit Prices
KAPA2G Fast DNA Polymerase
KK5008 KAPA2G Fast DNA Polymerase with dNTPs (100 U) 100 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM), dNTP Mix (10 mM) and extra MgCl2 (25 mM). *login for pricing
KK5009 KAPA2G Fast DNA Polymerase with dNTPs (250 U) 250 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM), dNTP Mix (10 mM) and extra MgCl2 (25 mM). *login for pricing
KK5020 KAPA2G Fast DNA Polymerase (100 U) 100 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM) and extra MgCl2 (25 mM). *login for pricing
KK5021 KAPA2G Fast DNA Polymerase (250 U) 250 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM) and extra MgCl2 (25 mM). *login for pricing
KK5101 KAPA2G Fast ReadyMix with dye (100 x 25 µL reactions) Kit includes 100 x 25 µl reactions of KAPA2G Fast ReadyMix with dye, a convenient 2X PCR master mix containing KAPA dNTPs, KAPA2G Fast DNA Polymerase, reaction buffer, Mg2+, and loading dye. *login for pricing
KK5102 KAPA2G Fast ReadyMix with dye (500 x 25 µL reactions) Kit includes 500 x 25 µl reactions of KAPA2G Fast ReadyMix with dye, a convenient 2X PCR master mix containing KAPA dNTPs, KAPA2G Fast DNA Polymerase, reaction buffer, Mg2+, and loading dye. *login for pricing
KAPA2G Fast HotStart DNA Polymerase
KK5500 KAPA2G Fast HotStart DNA Polymerase with dNTPs (500 U) 500 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM), dNTP Mix (10 mM) and extra MgCl2 (25 mM). *login for pricing
KK5501 KAPA2G Fast HotStart DNA Polymerase (500 U) 500 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM) and extra MgCl2 (25 mM). *login for pricing
KK5502 KAPA2G Fast HotStart DNA Polymerase with dNTPs (250 U) 250 U, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM), dNTP Mix (10 mM) and extra MgCl2 (25 mM). *login for pricing
KK5503 KAPA2G Fast HotStart DNA Polymerase (250 U) 250 units, 5 U/µL. Supplied with proprietary 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM) and extra MgCl2 (25 mM). *login for pricing
KK5510 KAPA2G Fast HotStart DNA Polymerase with dNTPs - Mg2+ free buffer (500 U) 500 units, 5 U/µL. Supplied with Mg2+ free 5X KAPA2G Buffer M, dNTP Mix (10 mM) and MgCl2 (25 mM). *login for pricing
KK5511 KAPA2G Fast HotStart DNA Polymerase - Mg2+ free buffer (500 U) 500 units, 5 U/µL. Supplied with Mg2+ free 5X KAPA2G Buffer M and MgCl2 (25 mM). *login for pricing
KK5512 KAPA2G Fast HotStart DNA Polymerase with dNTPs - Mg2+ free buffer (250 U) 250 units, 5 U/µL. Supplied with Mg2+ free 5X KAPA2G Buffer M, dNTP Mix (10 mM) and MgCl2 (25 mM). *login for pricing
KK5513 KAPA2G Fast HotStart DNA Polymerase - Mg2+ free buffer (250 U) KAPA2G Fast HotStart DNA Polymerase.^(250 U, 5 U/µL). With Mg-free proprietary 5X KAPA2G Buffer M and MgCl2 (25 mM). *login for pricing
KK5519 KAPA2G Fast HotStart DNA Polymerase (2,500 U) 2500 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM) and extra MgCl2 (25 mM). *login for pricing
KK5523 KAPA2G Fast HotStart DNA Polymerase (100 U) 100 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM) and extra MgCl2 (25 mM). *login for pricing
KK5530 KAPA2G Fast HotStart DNA Polymerase with dNTPs (100 U) 100 units, 5 U/µL. Supplied with 5X KAPA2G Buffer A (contains Mg2+ at a 1X conc. of 1.5 mM), dNTP Mix (10 mM) and extra MgCl2 (25 mM). *login for pricing
KK5601 KAPA2G Fast HotStart ReadyMix (500 x 25 µL rxns) KAPA2G Fast HotStart DNA Polymerase in a convenient 2X ReadyMix format. Contains Mg2+ at a 1X conc. of 1.5 mM. *login for pricing
KK5603 KAPA2G Fast HotStart ReadyMix (100 x 25 µL rxns) KAPA2G Fast HotStart DNA Polymerase in a convenient 2X ReadyMix format. Contains Mg2+ at a 1X conc. of 1.5 mM. *login for pricing
KK5608 KAPA2G Fast HotStart ReadyMix with dye (100 x 25 µL reactions) KAPA2G Fast HotStart DNA Polymerase in a convenient 2X ReadyMix format with loading dye. Contains Mg2+ at a 1X conc. of 1.5 mM. *login for pricing
KK5609 KAPA2G Fast HotStart ReadyMix with dye (500 x 25 µL reactions) KAPA2G Fast HotStart DNA Polymerase in a convenient 2X ReadyMix format with loading dye. Contains Mg2+ at a 1X conc. of 1.5 mM. *login for pricing

 

Fast PCR

Achieve high speed and sensitivity with the KAPA2G Fast DNA Polymerase. For more information download the new Fast PCR application note. Download here>>

Fast Multiplex PCR

The KAPA2G Fast DNA Polymerase is capable of fast multiplex PCR and increased sensitivity. For more information download the new Multiplex PCR application note. Download here>>


Product Description

KAPA2G Fast DNA Polymerase, a second-generation enzyme derived through a process of molecular evolution. KAPA2G Fast DNA Polymerase was specifically engineered for higher processivity and speed, offering significantly faster extension rates than wild-type Taq polymerase. 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 Fast HotStart is particularly well suited for high throughput Fast PCR, offering total reaction times 20 - 70% shorter than conventional PCR assays performed with wild-type Taq polymerase or hot start formulations thereof. In addition, KAPA2G Fast HotStart has been shown to achieve higher yields and sensitivity than competitor enzymes across a wide range of amplicon types. No specialized PCR consumables or thermocyclers are required.

KAPA2G Fast DNA Polymerase and optimized buffer system offers:

  • Extension times as low as 1 sec per kilobase.
  • Outstanding performance when compared to conventional hot start Taq polymerase.
  • 20 - 70% reductions in total PCR time.
  • No requirement for specialist PCR consumables or instrumentation.


Product Applications

Any standard, end-point PCR assay performed efficiently with wild-type Taq polymerase (or a hot start formulation thereof) may be converted to a Fast PCR assay with KAPA2G Fast HotStart. Amplicons generated with KAPA2G Fast HotStart are suitable for routine downstream applications, including restriction enzyme digestion, cloning and sequencing.


Product Performance

The ultimate in speed and performance

KAPA2G Fast HotStart and optimized buffer system is capable of dramatically reduced extension times and exceptional performance.

Hot start formulations of wild-type Taq do not perform satisfactorily when cycling times are reduced. In contrast, the unique properties of KAPA2G Fast HotStart allows for efficient amplification in total reaction times that are typically reduced by 40 - 70%.

Fast amplification of 7 human gene fragments using KAPA2G Fast HotStart or competitor hot start Taq formulations. Reactions (25 µl) contained 5 ng human genomic DNA and 0.5 units (KAPA2G Fast HotStart or Competitor I) or 0.625 units (Competitor A) enzyme. Cycling was performed on an EppendorfMastercyclerepgradient S, using a 3-step (35 cycle) profile with 15 sec denaturation (95ºC), 15 sec annealing (60ºC) and 1 sec extension (72ºC) per cycle. Initial denaturation/ enzyme re-activation time (95ºC) was 2 min for KAPA2G Fast HotStart and Competitor I and 7.5 min for Competitor A. The total reaction time was 29.5 min for KAPA2G Fast HotStart and Competitor I and 37 min for Competitor A.
The gene target, length and GC content of each amplicon is as follows: 1 = 5,10-methylene tetrahydrofolatereductase, 150 bp, 54.0% GC; 2 = zinc finger protein 638, 318 bp, 30.9% GC; 3 = attractin, 373 bp, 37.1% GC; 4 = zinc finger protein 41, 502 bp, 42.5% GC; 5 = PCI domain, 526 bp, 39.3% GC; 6 = chromosome 6 ORF167, 599 bp, 35.6% GC and 7 = epidermal growth factor receptor, 626 bp, 42.0% GC.


KAPA2G Fast HotStart outperforms hot start formulations of wild-type Taq, even when the latter are used at their optimal, slow extension rates (1 min/kb per cycle). The unique properties of this second-generation enzyme, combined with a proprietary HotStart antibody and optimized buffer system allows for efficient fast amplification of a larger variety of amplicon types than competitor enzymes. In the example below, competitor enzymes failed or performed poorly with 40 - 60% of a set of single copy human gene fragments, particularly in cases were amplicons are short or their GC content is high (>65%).

Amplification of 5 human gene fragments using KAPA2G Fast HotStart or competitor hot start Taq formulations. Reactions (25 µl) contained 5 ng human genomic DNA and 0.5 units (KAPA2G Fast HotStart or Competitor I) or 0.625 units (Competitor A or Competitor Q) enzyme. For amplicons with a GC content >65% (#2 and 3), 7.5% DMSO was included in reaction mixes. Cycling was performed on an EppendorfMastercyclerepgradient S, using 3-step cycling profiles (35 cycles) with 15 sec denaturation (95ºC) and 15 sec annealing (60ºC) per cycle for all enzymes. Extension (72ºC) was 1 sec per cycle for KAPA2G Fast HotStart and 60 sec per cycle for competitor enzymes. Initial denaturation/enzyme re-activation and final extension times used for each enzyme were as per the manufacturers recommendations. The total reaction time for each enzyme is as indicated.
The gene target, length and GC content of each amplicon is as follows: 1 = 5,10-methylene tetrahydrofolatereductase, 150 bp, 54.0% GC; 2 = guanine nucleotide binding protein, 241 bp, 83.8% GC; 3 = RET proto-oncogene, 392 bp, 79.1% GC; 4 = zinc finger protein 41, 502 bp, 42.5% GC and 5 = epidermal growth factor receptor, 626 bp, 42.0% GC.


High speed and sensitivity

KAPA2G Fast HotStart is based on a second-generation polymerase with the intrinsic ability to synthesize DNA faster than wild-type Taq and other DNA polymerases. Fast PCR protocols using KAPA2G Fast HotStart are primarily based on reduced extension times that allow for a significant reduction in PCR cycling time without the risk of compromising reaction performance.

Using KAPA2G Fast HotStart, a total extension time of 1 second per cycle is sufficient for the reproducible amplification of fragments <1 kb from as little as a single target copy of human genomic DNA. For amplicons >1 kb, 15 sec/kb extension time per cycle is used.

Amplification of a 626 bp fragment of the epidermal growth factor receptor (left) and a 1.3 kb fragment of the macrophage stimulating 1 receptor (right) from a 10-fold dilution series of human genomic DNA using KAPA2G Fast HotStart. 0.5 units of enzyme were used per 25 µl reaction. Cycling was performed on the G-Storm GS1 thermocycler with a fast block, using a standard 3-step cycling profile (35 cycles). For the 626 bp fragment, a total extension time of 1 sec per cycle was used and for the 1.3 kb fragment an extension time of 15 sec/kb (20 sec per cycle).


Fast, more sensitive Multiplex PCR

With KAPA2G Fast HotStart, faster Multiplex PCR is also possible. Because of the complexity of multiplex assays, longer annealing times are maintained, but extension times may be reduced to 10 sec per cycle for fragments <1 kb. Extension at 65ºC instead of 72ºC may also improve results. With optimized KAPA2G Fast HotStart protocols, time savings of 35 - 70% can be achieved.

Multiplex PCR assays typically employ a high template concentration. Using KAPA2G Fast HotStart in 1.5x KAPA2G HotStart buffer, improved sensitivity was achieved in all model systems tested.

Amplification of 7 fragments of the human Duchenne muscular dystrophy (DMD) locus in a Multiplex PCR using KAPA2G Fast HotStart or competitor hot start Taq formulations. Reactions (25 µl) contained 100 ng (lanes 1, 3 and 5) or 10 ng (lanes 2, 4 and 6) female human genomic DNA, 0.25 µM of each primer, 3 mM MgCl2 and 1 unit enzyme. KAPA2G Fast HotStart reactions were performed in 1.5x KAPA2G HotStart buffer A and competitor reactions 1x reaction buffer. Cycling was performed on an EppendorfMastercyclerepgradient S, using 3-step cycling profiles (30 cycles) and an initial denaturation/enzyme re-activation of 2 min for KAPA2G Fast HotStart and Competitor I and 10 min for Competitor A. For KAPA2G Fast HotStart, each cycle consisted of 15 sec denaturation (95ºC ), 30 sec annealing (55ºC) per cycle and extension for 10 sec at 72ºC. For competitor enzymes, each cycle consisted of 30 sec denaturation (95ºC), 30 sec annealing (55ºC) and extension for 45 sec at 72ºC. The total reaction time for each enzyme is as indicated.
Fragments amplified in this multiplex assay are (from top to bottom): exon Pm, 535 bp; exon 48, 506 bp; exon 19, 459 bp; exon 50, 271 bp; exon 13, 238 bp; exon 4, 196 bp and exon 47,181 bp.
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DocumentTypeDownload
KAPA2G Fast ReadyMix with dye Routine PCR Note Application Note Download
KAPA2G Fast Fast PCR Note Application Note Download
KAPA2G Fast HotStart Multiplex PCR Note Application Note Download
KAPA2G Fast ReadyMix MSDS Material Safety Data Sheet Download
KAPA2G Fast MSDS Material Safety Data Sheet Download
KAPA2G Fast HotStart ReadyMix MSDS Material Safety Data Sheet Download
KAPA2G Fast HotStart MSDS Material Safety Data Sheet Download
KAPA2G Fast Brochure Product Brochure Download
KAPA2G Fast TDS Technical Data Sheet Download
KAPA2G Fast HotStart ReadyMix TDS Technical Data Sheet Download
KAPA2G Fast HotStart TDS Technical Data Sheet Download
KAPA2G Fast ReadyMix TDS Technical Data Sheet Download


Frequently Asked Questions

KAPA2G Fast DNA Polymerase is an engineered enzyme and should not be evaluated using standard protocols for wild-type Taq. For optimal results, it is important to follow the recommendations provided in the KAPA2G Fast or KAPA2G Fast HotStart Technical Data Sheet. Fast PCR is not recommended for difficult amplicons (i.e. primer-template combinations that do not work well with wild-type Taq), or assays that have not been optimized.

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

  • Extension time is too long. Use 1 sec per cycle for amplicons ≤1 kb.
  • Annealing time is too long. Use 15 sec per cycle, or 10 sec per cycle for slow cyclers or reduced reaction volumes (<25 µl).
  • Suboptimal annealing temperature. Use an annealing temperature in the range of 55 – 65 °C, or perform an Ta gradient PCR (52 – 72 °C) to determine the optimal Ta for your assay.
  • Too much starting template. 10 – 25 ng genomic DNA or 1 – 10 ng plasmid/lambda DNA per 25 µl reaction should be sufficient for most applications.
  • Template quality is poor. Successful Fast PCR requires DNA that is not degraded or contaminated with inhibitors. Only use template DNA with an A260/A280 ratio between 1.7 and 2.0.
  • Too much enzyme. Use 0.5 U KAPA2G Fast per 25 µl reaction.
  • 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.
  • Genomic target is too long. Fragments up to 5 kb may be amplified from plasmid or lambda DNA with KAPA2G Fast, but fast amplification of genomic targets >3.5 kb is not recommended.
  • Reaction volumes are too large. Do not exceed reaction volumes of 25 µl.

2. What are the recommended applications for KAPA2G Fast PCR Kits?

  • Standard, end-point PCR assays normally performed with wild-type Taq (or hot start formulations thereof) – but completed in 20 – 70% less time.
  • High-throughput end-point PCR.
  • End-point PCR for genetic testing or downstream Sanger sequencing.
  • Fast Multiplex PCR.

3. Does it matter whether I use KAPA2G Fast or KAPA2G Fast HotStart?
KAPA2G Fast HotStart is an antibody-mediated hot start formulation of KAPA2G Fast DNA Polymerase. Both enzymes are supplied with a single, proprietary reaction buffer, specifically designed for Fast PCR. The non-HotStart and HotStart formulations should give similar results in most assays. However, for Multiplex PCR and workflows that require room temperature setup, the HotStart formulation must be used.

4. When should I order KAPA2G Fast HotStart ReadyMix?
KAPA2G Fast HotStart ReadyMix contains KAPA2G Fast HotStart DNA Polymerase (at an equivalent of 0.5 U per 25 µl reaction), 2x KAP2G Buffer A, 0.4 mM of each dNTP and 3 mM MgCl2 in an easy-to-use 2x ReadyMix format. It is recommended instead of the separate components supplied in KAPA2G Fast HotStart PCR Kits for any Fast PCR assay, except for Fast Multiplex PCR. It is the preferred product for high-throughput Fast PCR.

5. When should I order KAPA2G Fast ReadyMix with dye?

KAPA2G Fast ReadyMix with dye contains KAPA2G Fast DNA Polymerase (at an equivalent of 0.5 U per 25 µl reaction), 2X KAP2G Buffer A, 0.4 mM of each dNTP and 3 mM MgCl2 in an easy-to-use 2X ReadyMix format. It also contains loading dye, which allows for loading of PCR products in gels directly after PCR. It is recommended for routine PCR that does not require a HotStart formulation, and employs agarose gel electrophoresis as the method of analysis.

 

 

6. When should I order a KAPA2G Fast HotStart PCR Kit with Mg-free buffer?
KAPA2G Buffer A, the optimized buffer supplied with KAPA2G Fast and KAPA2G Fast HotStart PCR Kits, contains MgCl2 at a 1X concentration of 1.5 mM. KAPA2G Buffer M is only supplied in combination with the KAPA2G Fast HotStart enzyme. Buffer M is identical to Buffer A, with the exception that it does not contain Mg. Kits with Mg-free buffer are only recommended for Multiplex PCR applications.

 

 

7. Can I store KAPA2G Fast PCR Kits at room temperature or 4 °C?
The recommended temperature for long-term storage of KAPA2G Fast enzymes, KAPA2G Buffer A, 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!)

8. What is Fast PCR?
Fast PCR is PCR in which total reaction time is reduced by reducing the duration of one or more of the steps during each cycle of a PCR. Using the KAPA2G Fast PCR Kit, PCR time can be reduced by 20 – 70% by simply reducing the extension time in each cycle. Additional time savings may be achieved by optimizing other cycling parameters (e.g. denaturation and/or annealing time) for your specific assay and thermocycler.

9. Why is the KAPA2G Fast PCR Kit the preferred product for Fast PCR?
KAPA2G Fast PCR Kits are unique they contain the KAPA2G Fast DNA Polymerase, a second-generation enzyme that is capable of synthesizing DNA much faster than Taq and other DNA polymerases. This means that reaction times can be reduced significantly by simply reducing the extension time in each cycle. With KAPA2G Fast PCR Kits, high performance, high speed PCR is possible without the need for extensive optimization, special PCR consumables or specialist thermocyclers. In contrast, competitor kits based on wild-type Taq polymerase are limited by the extension rate of Taq, and timesaving rely primarily on the reduction of denaturation and annealing times. Such “artificially” shortened protocols often result in reduced reaction efficiency, especially with longer amplicons, lower target copy numbers and faster thermocyclers.

10. Can any existing PCR assay be converted to a Fast PCR assay?
Any standard, single-amplicon end-point PCR assay may be converted to a Fast assay, provided that reaction volumes do not exceed 25 µl and a sufficient amount of good quality target DNA is used. The KAPA2G Fast PCR Kit is validated for Fast amplification of fragments up to 5 kb from simple (e.g. plasmid or lambda) DNA, and up to 3.5 kb from complex (e.g. genomic) DNA. Fast PCR is not recommended for PCR assays that do not work well with wild-type Taq, PCR assays that have not been optimized, primer-template combinations that produce low yields of specific product, even after extensive optimization, low-efficiency PCR assays, or advanced PCR applications such as fingerprinting PCR, mutagenesis PCR or PCR involving the incorporation of nucleotide analogs.

11. Is Fast Multiplex PCR possible?
Existing Multiplex PCR assays can be converted to Fast Multiplex PCR assays with KAPA2G Fast HotStart. Optimal reaction parameters for Fast Multiplex PCR are slightly different to those recommended for standard, single-amplicon assays. Please refer to the KAPA2G Fast HotStart Multiplex PCR Application Note for full details of reaction conditions and optimization strategies for Fast Multiplex PCR. KAPA2G Fast HotStart PCR Kits with Mg-free buffer is recommended for Multiplex PCR applications, as this facilitates the optimization of Mg concentration.

12. Do I have to buy a special thermocycler to do Fast PCR?
KAPA2G Fast PCR Kits may be used for Fast PCR on any conventional (Peltier-based) thermocycler with a block, together with standard thin-walled PCR tubes or plates. Using this kit, a significant amount of PCR time may be saved irrespective of whether you have a slow or fast cycler. For optimal performance, it is important to know the approximate ramping (heating and cooling) rates of your specific instrument. Total reaction times on fast cyclers are shorter, but the times programmed for each step of the PCR may have to be longer than for a slow cycler to ensure equal performance (yield/sensitivity). This is because slow cyclers offer “extra time” that contributes to denaturation and annealing because they take longer to heat and cool the block between consecutive steps and cycles.

13. What are the recommended extension rates for KAPA2G Fast DNA Polymerase?
The recommended extension rate depends on amplicon length. For amplicons ≤1 kb, use 1 sec extension time per cycle. This may be increased to a maximum of 5 sec per cycle to improve yields. For amplicons >1 kb, use an extension time of 15 sec/kb per cycle. Too long extension times are likely to result in non-specific amplification and/or smearing.

14. What is the optimal annealing time for Fast PCR?
Annealing time is a critical parameter in successful Fast PCR using KAPA2G Fast enzymes. Annealing times that are too short may lead to low yields or reaction failure, whereas too annealing times that are too long, is likely to generate non-specific amplicons or smears. Never use more than 15 sec annealing time per cycle. For small reaction volumes (<25 µl) or slow thermocyclers, this may be reduced to 10 sec per cycle.

15. What is the optimal annealing temperature for Fast PCR?
The optimal annealing temperature for any PCR assay depends on the specific template-primer combination. Primers for Fast PCR assays should be designed for use at an annealing temperature between 55 and 65 °C. KAPA2G Buffer A, the optimized buffer for Fast PCR, is designed to facilitate specific primer annealing over a wider range of annealing temperatures than other PCR buffers. When converting an existing assay to a Fast PCR assay, start with the same annealing temperature as used with wild-type Taq as a first approach. To obtain the highest yield of specific product with KAPA2G Fast PCR Kits, perform an annealing temperature gradient PCR in the range of 52 – 72 °C.

16. Should I convert to a 2-step profile to save time?
2-step profiles are only suitable for primer pairs that anneal effectively above 65 °C or across the range of 65 – 72 °C and are not recommended for existing primer pairs with sharply defined optimal annealing temperatures <65 °C. Converting from a 3-step to a 2-step assay with unsuitable primers is likely to require extensive optimization to reduce reaction time without compromising reaction performance, especially on fast ramping thermocyclers. A significant amount of time can be saved by converting an existing 3-step assay to a Fast 3-step assay by simply applying the unique, fast extension rates of the KAPA2G Fast DNA Polymerase without extensive optimization or compromising reaction performance.

17. Why is reaction volume important for successful Fast PCR?
Efficient denaturation is important for reaction performance and is dependent on efficient heat transfer to reaction components. Heat transfer becomes less efficient as reaction volume is increased. Since the temperature inside the reaction tube lags behind the temperature of the thermocycler block – and this lag is bigger on fast cyclers than on slow cyclers – it is important to ensure optimal heat transfer. Always use the smallest reaction volume (always <25 µl), thin-walled PCR tubes or plates that fit the thermocycler block well, and sufficient denaturation times. The above is particularly important for some templates, e.g. those with a high GC content or stable secondary structure, which are more difficult to denature.

18. What is the optimal Mg concentration for Fast PCR?
The optimal Mg concentration for any PCR assay depends on the specific combination of template and primers. A final Mg concentration of 1.5 mM is sufficient for the majority of Fast PCR assays. However, if you are converting an existing assay that requires >1.5 mM Mg to a Fast assay, it is important that the same final concentration of Mg is used.

19. Are KAPA2G Fast enzymes compatible with PCR additives?
KAPA2G Fast PCR Kits are not recommended for assays that require additives, as these are typically “difficult”. KAPA2G Robust PCR Kits are recommended instead. However, KAPA2G Fast enzymes may be used for assays performed successfully with wild-type Taq in the presence of DMSO at a final concentration ≤5%.

20. Can I still use KAPA2G DNA Polymerase if my existing assay requires a specialized buffer?
KAPA2G Buffer A, supplied in KAPA2G Fast and Fast HotStart PCR Kits, is a proprietary buffer designed specifically for Fast PCR. For optimal results with KAPA2G Fast enzymes, it is highly recommended that this buffer is used. KAPA2G Fast enzymes should be compatible with any standard PCR buffer developed for use with wild-type or hot start Taq, provided that the pH is 8.3 or higher. Optimal performance in specialized buffers is not guaranteed and it is likely that annealing temperature and time, as well as extension time will have to be determined empirically.

21. Can PCR products generated with KAPA2G Fast PCR Kits be digested, cloned and sequenced?
Yes, PCR products generated with KAPA2G Fast enzymes have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3’-dA-tailed and may be used for TA cloning, or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, purification of PCR products using any standard PCR cleanup kit is recommended.

22. Can PCR products generated with KAPA2G Fast PCR Kits be analysed by dHPLC?
Yes. PCR products generated with KAPA2G Fast or KAPA2G Fast HotStart, using KAPA2G Buffer A or M 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 KAPA2G Fast HotStart ReadyMix must be diluted >2.5 times, or purified using a standard PCR cleanup kit, prior to analysis on Transgenomic WAVE dHPLC systems.

23. What is the loading dye contained in KAPA2G Fast ReadyMix with dye?

KAPA2G Fast ReadyMix with dye contains a mixture of two dye components, which gives the ReadyMix a green colour at the time of loading. This resolves into a blue and an orange component during electrophoresis. The blue band migrates with fragments of 3 - 4 kb in a 1% gel, whereas the orange band migrates with fragments <500 bp.