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  • Protocol for OneTaq® 2X Master Mix with Standard Buffer (M0482)

    Overview

    PCR

    The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1). Taq DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using New England Biolabs’ OneTaq 2X Master Mix with Standard Buffer. 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: 

    We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C).

    Add to a sterile thin-walled PCR tube:
    Component 25 μl reaction 50 μl reaction Final Concentration
    10 µM Forward Primer 0.5 µl 1 μl 0.2 µM
    10 µM Reverse Primer 0.5 µl 1 μl 0.2 µM
    Template DNA variable variable < 1,000 ng
    OneTaq 2X Master
    Mix with Standard Buffer
    12.5 µl 25 μl 1X
    Nuclease-free water to 25 µl to 50 µl < 1,000 ng
    Notes: Gently mix the reaction. 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
    45-68°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 Primer3 can be used to design or analyze primers. The final concentration of each primer in a PCR may be 0.05–1 µM, typically 0.2 µM

    3. Mg++ and Additives:

      Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with OneTaq DNA Polymerase. The final Mg++ concentration in 1X OneTaq Master Mix with Standard Buffer is 1.8 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.2 mM increments using MgCl2 (NEB# B9021).

      For amplification of difficult targets, like GC-rich sequences, we recommend OneTaq 2X Master Mix with GC Buffer (NEB# M0483). Alternatively, DMSO or formamide may be used

    4. Denaturation:

      An initial denaturation of 30 seconds at 94°C is sufficient to amplify most targets from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2–4 minutes at 94°C is recommended prior to PCR cycling to fully denature the template. Alternatively, use OneTaq Hot Start 2X Master Mix with GC Buffer. With colony PCR, an initial 2–5 minute denaturation at 94°C is recommended to lyse cells.

      During thermocycling a 15–30 second denaturation at 94°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 temperature for PCR.

    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. PCR Product:

      The majority of the PCR products generated using OneTaq 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.