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  • Protocol for OneTaq® Hot Start 2X Master Mix with GC Buffer (M0485)

    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 GC Buffer. These guidelines cover routine PCR. Specialized applications may require further optimization.

    Introduction

    Reaction setup: 

    Due to the presence of the inhibitor, 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.

    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 Hot Start 2X
    Master Mix with
    GC Buffer
    12.5 μl 25 μl 1X
    OneTaq High
    GC Enhancer,
    (optional)*
    (2.5–5 μl) (5–10 μl) (10–20%)
    Nuclease-free water to 25 µl to 50 µl  
    *For extremely difficult or high GC amplicons, the addition of 10–20% OneTaq High GC Enhancer may improve amplification.

    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: 

    Initial denaturation: 
    94°C   30 seconds

    30 cycles:
    94°C   15-30 seconds  
    45–68°C   15-60 seconds
    68°C   1 minute per kb

    Final extension: 
    68°C   5 minutes 

    Hold: 4–10°C

    Protocol

    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 Hot Start Master Mix with GC Buffer is 2 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.2 mM increments using MgSO4 (NEB# B1003 ).


    4. Denaturation:

      No separate activation step is required to release the hot start inhibitor from the enzyme. An initial denaturation of 30 seconds at 94°C is sufficient to amplify most targets from pure DNA templates. For difficult templates, a longer denaturation of 2–4 minutes at 94°C is recommended prior to PCR cycling to fully denature the template. 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 yields sufficient product. Up to 45 cycles may be required to detect low copy number targets.

    8. 2-step PCR:

      When primers with annealing temperatures of 68°C or above are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.

    9. 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.