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

Materials Required but not Supplied

OneTaq® 2X Master Mix with Standard Buffer

Overview

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: 

  1. Assemble all reaction components. Each component should be gently mixed before adding to the reaction in a sterile thin-walled PCR tube. The entire reaction should be mixed again to ensure homogeneity. Collect all liquid to the bottom of the tube with a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

    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  
  2. Transfer PCR tubes from ice to a PCR machine with the block preheated to 94°C 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  
    *Use of the NEB TmCalculator is highly recommended.


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–10 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 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 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:
    A significant portion 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.

    The Monarch® Spin PCR & DNA Cleanup Kit (5 μg) (NEB #T1130) is recommended as an efficient method for purification and concentration up to 5 μg of high-quality, double-stranded and single-stranded DNA.

 

 

References:
1.  Saiki,R.K. et al (1985). Science. 230, 1350-1354.
2.  Powell,L.M. et. al. (1987). Cell. 50, 831-840.

 

 

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