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  • Protocol for a PCR reaction using Hot Start Taq 2X Master Mix (M0496)

    Introduction

    PCR

    The following guidelines are provided to ensure successful PCR using New England Biolabs’ Hot Start Taq 2X Master Mix. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization.


    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 to release the inhibitor from the enzyme.

    Add to a sterile thin-walled PCR tube:
    Component 25 μl reaction 50μl reaction Final Concentration
    10 µM Forward Primer 0.5 µl 1 μl 0.2 μM (0.05–1 μM)
    10 µM Reverse Primer 0.5 µl 1 μl 0.2 μM (0.05–1 μM)
    Template DNA variable variable < 1,000 ng
    Hot Start Taq 2X
    Master Mix
    12.5 μl 25 μl 1X
    Nuclease-free water to 25 µl to 50 µl  
    Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.

    Transfer PCR tubes to a PCR machine and begin thermocycling:

    Thermocycling conditions for a routine PCR: 

     
    STEP
    TEMP
    TIME
    Initial Denaturation
    95°C
    30 seconds
    30 Cycles 95°C
    45-68°C
    68°C
    15-30 seconds
    15-60 seconds
    1 minute/kb
    Final Extension 68°C
    5 minutes
    Hold 4-10°C


    General Guidelines: 
    1. Template:

      Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 μl reaction are as follows:
      DNA Amount
      Genomic 1 ng–1 µg
      Plasmid or viral 1 pg–1 ng

    2. Primers:

      Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as PrimerSelect™ and Primer 3 can be used to design or analyze primers. The final concentration of each primer in a PCR 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 Hot Start Taq DNA Polymerase. The final Mg++ concentration in 1X Hot Start Taq Master Mix is 1.5 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2 (NEB# B9021). 

      Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (1) or formamide (2).

    4. Denaturation:

      No separate activation step is required to release the hot start inhibitor from the enzyme. An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended. 

      During thermocycling a 15–30 second denaturation at 95°C is recommended.

    5. Annealing:

      The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°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 appropriate annealing temperatures for PCR.

      When primers with annealing temperatures above 68°C are used, a 2-step PCR protocol is possible (see #8).

    6. Extension:

      The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.

    7. Cycle number:

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

    8. 2-step PCR:

      When primers with annealing temperatures above 68°C are used, a 2-step thermocycling protocol is possible.

      Thermocycling Conditions for a Routine 2-Step PCR:
      STEP TEMP TIME
      Initial Denaturation 95°C 30 seconds
      30 Cycles 95°C 15–30 seconds
      68°C 1 minute/kb
      Final Extension 68°C 5 minutes
      Hold 4-10°C  

    9. PCR product: 

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

    References:
    1.  Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
    2.  Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res.. 18, 7465.