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  • PCR Protocol for LongAmp™ Hot StartTaq DNA Polymerase (M0534)

    Overview

    PCR

    The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique of DNA amplification (1). Taq DNA Polymerase is an enzyme widely used in PCR (2). LongAmp Hot Start Taq DNA Polymerase allows for greater PCR sensitivity and permits room temperature PCR set-up. The following guidelines are provided to ensure successful PCR using New England Biolabs’ LongAmp Hot Start Taq DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure or low template concentrations may require further optimization.

    Protocol

    Reaction setup: 

    Due to the hot-start nature of the enzyme, reactions can be assembled on the bench at room temperature and transferred to a thermocycler. No separate activation step is required.

    Component 25 µl reaction 50 µl reaction Final Concentration
    5X LongAmp Taq Reaction Buffer 5 µl 10 µl 1X
    10 mM dNTPs 0.75 µl 1.5 µl 300 µM
    10 µM Forward Primer 1 µl 2 µl 0.4 µM (0.05–1 µM)
    10 µM Reverse Primer 1 µl 2 µl 0.4 µM (0.05–1 µM)
    Template DNA variable variable <1,000 ng
    LongAmp Hot Start Taq DNA Polymerase 1 µl 2 µl 5 units/50 µl PCR
    Nuclease-free water to 25 µl to 50 µl  
    Notes: Gently mix the reaction. Avoid pipetting samples containing target DNA when amplicons above 20 kb are desired. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

    Transfer PCR tubes to a PCR machine and begin thermocycling.

    Thermocycling conditions for a routine PCR: 

    STEP
    TEMP
    TIME
    Initial Denaturation
    94°C
    30 seconds
    30 Cycles 94°C
    45-65°C
    65°C
    15-30 seconds
    15-60 seconds
    50 seconds/kb
    Final Extension 65°C
    10 minutes
    Hold 4-10°C
     

    General Guidelines:
     
    1. Template:

      The quality of the DNA template is essential for long-range PCR amplification. Recommended amounts of DNA template for a 50 μl reaction are as follows:
      DNA Up to 15 kb Above 15 kb
      Genomic 1 ng–500 ng 10 ng–1 µg
      Plasmid or Viral 1 pg–1 ng 10 pg–10 ng

      Successful amplification above 20 kb largely depends on the quality of DNA templates and the primer sequences.

    2. Primers: 

      Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (http://frodo.wi.mit.edu/primer3) can be used to design or analyze primers. For amplicons larger than 20 kb, it is desirable to have primers with GC content above 50%, matched Tm above 60°C and at least 24 nucleotides in length. The final concentration of each primer in a PCR reaction may be 0.05–1 μM, typically 0.1–0.5 μM.

    3. Mg++ and additives: 

      Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with LongAmp Hot Start Taq DNA Polymerase. The final Mg++ concentration in 1X LongAmp Taq Reaction Buffer is 2 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgSO4 (NEB# B1003). 

      Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4).

    4. Deoxynucleotides

      The recommended final concentration of dNTPs for long-range PCR is 300 μM of each deoxynucleotide.

    5. LongAmp Hot Start Taq DNA Polymerase concentration: 

      We generally recommend using LongAmp Hot Start Taq DNA Polymerase at a concentration of 100 units/ml (5 units/50 μl reaction). However, the optimal concentration of LongAmp Hot Start Taq DNA Polymerase may vary in specialized applications.

    6. Denaturation:

      LongAmp Hot Start Taq DNA Polymerase does not require a separate activation step.

      An initial denaturation of 30 seconds at 94°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2–4 minutes at 94°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 94°C is recommended. 

      During thermocycling, a 10-30 second denaturation at 94 °C is recommended.

    7. Annealing: 

      The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–65°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm. We recommend using NEB's Tm Calculator to determine an appropriate annealing temperature for PCR.

      When primers with annealing temperatures above 60°C are used, a 2-step PCR protocol is possible (see #10).

    8. Extension: 

      The recommended extension temperature is 65°C. Extension times are generally 50 seconds per kb. A final extension of 10 minutes at 65°C is recommended.

    9. Cycle number: 

      Generally, 25–35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.

    10. 2-step PCR: 

      When primers with annealing temperatures above 60°C are used, a 2-step thermocycling protocol is possible. 

      Thermocycling conditions for a routine 2-step PCR:
      STEP
      TEMP
      TIME
      Initial Denaturation 94°C 30 seconds
      30 Cycles 94°C
      60-65°C
      10-30 seconds
      50 seconds/kb
      Final Extension 60-65°C 10 minutes
      Hold 4-10°C
    11. PCR product:

      The majority of the PCR products generated using LongAmp Taq DNA Polymerase contain dA overhangs at the 3´–end; therefore the PCR products can be ligated to dT/dU-overhang vectors.

    References:
    1.  Saiki R.K. et al. (1985). Science. 230, 1350-1354.
    2.  Powell, L.M. et al. (1987). Cell. 50, 831-840.
    3.  Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
    4.  Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res. 18, 7465.