Introduction

The SequalPrep™ Long PCR Kit with dNTPs provides a robust system for long-range, high-fidelity PCR for use in next-generation sequencing applications. The kit includes all components for PCR except template, primers and water. With this system, little to no optimization is required to amplify targets up to 15 kb in length. SequalPrep™ Long Polymerase offers an automatic hot-start for simplified reaction assembly, and efficient and robust amplification of most non-optimized targets without a loss in fidelity.

Using the kit, you can develop an optimal formulation for your targets of interest by selecting from two separate enhancers. SequalPrep™ Long Polymerase is supplied at 5 units/μl, and is used at 1.8 units per 20-μl reaction. The 10X Reaction Buffer includes Mg++ and dNTPs at an optimal concentration.

Component Amount
SequalPrep™ Long Polymerase, 5 U/μl 200 μl
SequalPrep™ 10X Reaction Buffer 1.2 ml
SequalPrep™ 10X Enhancer A 1.2 ml
SequalPrep™ 10X Enhancer B 1.2 ml
DMSO 750 μl

Storage

Store all components at –20°C, except DMSO , which may be stored at 4°C or room temperature after initial  hawing. For long-term storage, refreeze DMSO.

Quality Control

The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available here, and is searchable by product lot number, which is printed on each box.

PCR Assembly

General PCR parameters and troubleshooting information are documented in Innis, et al (Innis et al., 1990). PCR reactions should be assembled in a DNA-free environment using clean, dedicated automatic pipettors and aerosol resistant barrier tips. Always keep the control DNA and other templates to be amplified isolated from the other components.

Recommendations and Guidelines

  • Reagents: Avoid freeze/thawing of primers and SequalPrep™ 10X Reaction Buffer by preparing single-use aliquots.
  • Primers: Optimal primer design is essential for long-range PCR. Primers should be 18–35 nucleotides in length, with a GC content of 45–65%. We recommend designing primers with at least 2 Cs or Gs at the 3’ end of the primer. Primers should be screened to avoid a high degree of similarity with other sequences in the genome, and to avoid primer dimers.
  • Template: Intact, high-quality genomic DNA is essential for longrange PCR. Do not vortex and avoid freeze/thawing of DNA. DNA should be free of PCR inhibitors and diluted in PCR-grade water, not TE (EDTA will chelate the Mg++ in the reaction buffer).
  • Enhancers: Two different PCR enhancers are provided at 10X concentration each. As a general starting point, we recommend using Enhancer A at a 0.5X final concentration. For targets that do not amplify, test Enhancer A at 1X and Enhancer B at 0.5X and 1X to determine the optimal enhancer/concentration.
  • Reaction Assembly: Reactions may be set up at room temperature.
  • Annealing Temperature: The initial annealing temperature is ~5° lower than the Tm of the primers. For higher specificity, it may be necessary to increase the annealing temperature in steps of 2°C.
  • Cycling Conditions: For higher yields, increase the total number of cycles to 40. For higher specificity of robust products, use a total of 30–35 cycles.
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Long-Range PCR Protocol

  1. Program the thermal cycler as follows (note that the annealing temperature will vary depending on the Tm of your primers):

    94°C                                 2 minutes

    10 cycles of:
    94°C                               10 seconds
    Primer Tm –5°C              30 seconds
    68°C                                1 minute/Kb

    20–30 cycles of:
    94°C                                10 seconds
    Primer Tm –5°C              30 seconds
    68°C                                1 minute/Kb (+20 sec/cycle)

    Final extension:
    72°C                                 5 minutes
    Hold at 4°C

  2. Prepare a master mix as follows. Amounts below are for a 20-μl reaction. Scale the reaction volume and multiply by number of reactions as needed.

    Component Volume Final Concentration
    SequalPrep™ 10X Reaction Buffer 2 μl 1X
    DMSO0.4 μl --
    SequalPrep™ 10X Enhancer A or B 1–2 μl* 0.5X–1X
    SequalPrep™ Long Polymerase, 5 U/μl0.36 μl1.8 U
    DNase-free waterto 18 μl


    *Use Enhancer A at 0.5X as a starting point. For targets that do not amplify, test with Enhancer A at 1X and Enhancer B at 0.5X and 1X.

  3. Pipette 18 μl of master mix into each PCR tube/well, and add:

    Primer mix (10 μM each)                1 μl                     0.5 μM each
    Template DNA (1–100 ng/μl)          1 μl                     as required

  4. Cap the tube/well, tap gently to mix, and centrifuge briefly.

  5. Place the tube in the thermal cycler and run the program from Step 1. After cycling, samples can be stored at –20°C until use.

  6. Analyze the amplification products by agarose gel electrophoresis. We recommend using E-Gel® 0.8% or 1.0% gels and the TrackIt™ 1 kb Plus DNA Ladder or 1 Kb DNA Extension Ladder (see Additional Products in ordering table).
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Reference

  1. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (eds) (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA
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MAN0000752         11-Jun-2010