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适用说明:本文档适用于KAPA HiFi HotStart ReadyMix PCR Kit (KK2631)。
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Product Description

KAPA HiFi HotStart DNA Polymerase is a novel B-family DNA polymerase, engineered to have increased affinity for DNA, without the need for accessory proteins or DNA binding domains. The intrinsic high processivity of the enzyme results in significant improvement in yield, speed and sensitivity when compared with wild-type B-family DNA polymerases. In addition, the ability to amplify long targets, as well as GC- and AT-rich targets, is significantly improved. The enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step. This prevents nonspecific amplification during reaction setup, increases sensitivity, and improves reaction efficiency.

In the ReadyMix PCR Kit, KAPA HiFi HotStart DNA Polymerase is supplied in a convenient 2X ReadyMix format, containing all reaction components except primers and template. The ReadyMix contains KAPA HiFi HotStart DNA Polymerase (0.5 U per 25 μL reaction) in a proprietary reaction buffer containing dNTPs (0.3 mM of each dNTP at 1X), MgCl2 (2.5 mM at 1X) and stabilizers.

KAPA HiFi HotStart ReadyMix is designed for routine, high-fidelity PCR of a wide range of targets and fragment sizes. It offers error rates approximately 100 times lower than wild-type Taq DNA polymerase, and higher success rates and yields than achievable with wild-type B-family (proofreading) DNA polymerases. In addition, KAPA HiFi HotStart requires significantly shorter reaction times than wild-type B-family DNA polymerases.

KAPA HiFi HotStart DNA Polymerase has 5’→3’ polymerase and 3’→5’ exonuclease (proofreading) activity, but no 5’→3’ exonuclease activity. The strong 3’→5’ exonuclease activity results in superior accuracy during DNA amplification, lending to KAPA HiFi HotStart DNA Polymerase the lowest published error rate of all B-family DNA polymerases (1 error per 3.6 x 106 nucleotides incorporated). This fidelity is approximately 100 times higher than that of wild-type Taq DNA polymerase, and up to 10 times higher than that of other B-family DNA polymerases and polymerase blends.

DNA fragments generated with KAPA HiFi HotStart ReadyMix may be used for routine downstream analysis and applications, including restriction enzyme digestion, cloning and sequencing. PCR products generated with KAPA HiFi HotStart ReadyMix are blunt-ended, but may be 3’-dA-tailed for cloning into TA cloning vectors (see Important Parameters: TA cloning).

Product Applications

The KAPA HiFi HotStart PCR Kit is ideally suited for:
• PCR for conventional sequencing (direct sequencing or sequencing of cloned PCR products)
• Next-generation sequencing library amplification (when used in conjunction with the KAPA Library Amplification protocol)
• Amplification of DNA fragments for cloning and protein expression or genomic characterization
• Site-directed mutagenesis.
For more information on these and other high-fidelity PCR applications, please refer to the KAPA HiFi Application Notes on Site-Directed Mutagenesis, Routine High-Fidelity PCR, and High-Fidelity GC-rich PCR available from www.kapabiosystems.com.

Standard PCR Protocol

IMPORTANT! The KAPA HiFi HotStart ReadyMix contains an engineered B-family (proofreading) DNA polymerase and uniquely-formulated buffers, and requires specialized reaction conditions. If these conditions are not adhered to, reaction failure is likely. Refer to Important Parameters for more information.

Step 1: Prepare the PCR master mix
• KAPA HiFi HotStart reactions MUST be set up on ice since the high proofreading activity of the enzyme will result in rapid primer degradation at room temperature.
• Ensure that all reagents are properly thawed and mixed.
• Prepare a PCR master mix containing the appropriate volume of all reaction components common to all or a subset of reactions to be performed.
• Calculate the required volumes of each component based on the following table:

1 Reaction volumes may be adjusted between 10–50 μL. For volumes other than 25 μL, scale reagents proportionally. Reaction volumes > 50 μL are not recommended.
2 When using for next-generation sequencing library amplification, please use protocol provided with the KAPA Library Amplification Kit.
3 KAPA HiFi HotStart ReadyMix contains 2.5 mM MgCl2 (1X). Additional MgCl2 may be added separately, but is unlikely to be required.
4 KAPA HiFi HotStart ReadyMix contains 0.3 mM of each dNTP (1X), and 0.5 U of KAPA HiFi HotStart DNA Polymerase (per 25 μL reaction) in a proprietary reaction buffer.
5 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
• Transfer the appropriate volumes of PCR master mix, template and primer to individual PCR tubes or wells of a PCR plate.
• Cap or seal individual reactions, mix and centrifuge briefly.

Step 3: Run the PCR
• Perform PCR with the following cycling protocol1:

1 When using for next-generation sequencing library amplification, please use protocol provided with the KAPA Library Amplification Kit.
2 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).
3 KAPA HiFi HotStart ReadyMix has a higher salt concentration than conventional PCR ready-mixes, which affects DNA melting. To ensure that complex and GC rich targets are completely denatured, use a temperature of 98 °C for denaturation during cycling.
4 In addition to DNA melting, high salt also affects primer annealing. The optimal annealing temperature for a specific primer set is likely to be different (higher) than when used in a conventional PCR ready-mix. An annealing temperature gradient PCR is recommended to determine the optimal annealing temperature with the KAPA HiFi HotStart ReadyMix. If gradient PCR is not feasible, anneal at 65 °C as a first approach.
5 Two-step cycling protocols with a combined annealing/extension temperature in the range of 68–75 °C and a combined annealing/extension time of 30 sec/kb may be used.
6 Use 15 sec extension per cycle for targets ≤ 1 kb, and 30–60 sec/kb for longer fragments, or to improve yields.
7 For highest fidelity, use ≤ 25 cycles. In cases where very low template concentrations or low reaction efficiency results in low yields, 30–35 cycles may be performed to produce sufficient product for downstream applications.

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