In this protocol

适用说明:本说明书适用于KAPA HiFi HotStart PCR Kit (KK2500, KK2501及KK2502)。
友情提示:本网页说明书摘取了原说明书中的产品描述,产品应用及实验操作流程、故障排查等主要信息,欲了解关于更多信息,请参考原说明书 (点击面板上“下载PDF”可下载)。

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 to 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.

KAPA HiFi HotStart PCR Kits are designed for routine, high-fidelity PCR of a wide range of targets and fragment sizes. They offer error rates approximately 100 times lower than wild-type Taq DNA polymerase, and higher success 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 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 DNA Polymerase may be used for routine downstream analysis and applications, including restriction enzyme digestion, cloning and sequencing. PCR products generated with KAPA HiFi HotStart PCR Kits are blunt-ended, but may be 3’-dA-tailed for cloning into TA cloning vectors (see Important Parameters: TA cloning).

KAPA HiFi DNA Polymerase is supplied with two uniquely-formulated PCR buffers for optimal performance. Both buffers contain MgCl2 at a 1X concentration of 2 mM. The Fidelity Buffer is recommended for routine high-fidelity PCR, while the GC Buffer is recommended for the amplification of GC-rich and other difficult targets. The additives in the GC Buffer result in a 2-fold decrease in fidelity when compared with the Fidelity Buffer.

Product Applications

The KAPA HiFi HotStart PCR Kit is ideally suited for:
• PCR for conventional sequencing (direct sequencing or sequencing of cloned PCR products)
• 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.sequencing.roche.com.

Standard PCR Protocol

IMPORTANT! The KAPA HiFi HotStart PCR Kit 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 down proportionally. Reaction volumes > 50 μL are not recommended.
2 KAPA HiFi Buffers contain 2 mM MgCl2 (1X). Additional MgCl2 may be added separately. Use the GC Buffer only if the Fidelity Buffer gives poor results.
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.

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 protocol:

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).
2 KAPA HiFi Buffers have a higher salt concentration than conventional PCR buffers, 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.
3 In addition to DNA melting, the high-salt buffers also affect primer annealing. The optimal annealing temperature for a specific primer set is likely to be different (higher) than when used in a conventional PCR buffer. An annealing temperature gradient PCR is recommended to determine the optimal annealing temperature with KAPA HiFi HotStart. If gradient PCR is not feasible, anneal at 65 °C as a first approach.
4 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.
5 Use 15 sec extension per cycle for targets ≤ 1 kb, and 30-60 sec/kb for longer fragments, or to improve yields.
6 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.

Troubleshooting

  • Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
    Q&A

    Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
    You are highly recommended to post your data including images for the troubleshooting.

    You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.