Protocol for Hot Start Taq DNA Polymerase with Standard Taq Reaction Buffer (M0534)

Overview

PCR

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification. Taq DNA Polymerase is an enzyme widely used in PCR. Hot Start Taq DNA Polymerase allows for greater amplification sensitivity and increased ease of reaction setup. The following guidelines are provided to ensure successful PCR using New England Biolabs’ Hot Start Taq DNA Polymerase. These guidelines cover routine PCR reactions. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb 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 to release the inhibitor from the enzyme.

Add to a sterile thin-walled PCR tube:

Component 25 μl reaction 50 μl reaction Final Concentration
10X Standard Taq
Reaction Buffer
2.5 µl 5 μl 1X
10 mM dNTPs 0.5 µl 1 μl 200 mM
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)
Hot Start Taq DNA
Polymerase
0.125 µl 0.25 μl 1.25 units/50 µl PCR
Template DNA variable variable <1,000 ng
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. 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
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 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 Hot Start Taq DNA Polymerase. The final Mg++ concentration in 1X ThermoPol Reaction Buffer is 2.0 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 (1) or formamide (2).

  4. Deoxynucleotides:

    The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.

  5. Hot Start Taq DNA Polymerase Concentration:

    We generally recommend using Hot Start Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of Hot Start Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 μl reaction) in specialized applications.

  6. Denaturation:

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

    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 incubation at 95°C is recommended to lyse cells.

    During thermocycling a 15–30 second denaturation at 95°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 appropriate annealing temperature for PCR.


    When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible.

  8. 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.

  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 65°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
    65-68°C
    15-30 seconds
    1 minute/kb
    Final Extension 65-68°C 5 minutes
    Hold 4-10°C
  11. 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.