PCR Protocol for Crimson Taq DNA Polymerase with (Mg-free) Buffer (M0325)

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 NEB’s Crimson Taq DNA Polymerase. 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.

Protocol

Reaction setup: 
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).

Component 25 μl reaction 50 μl reaction Final Concentration
5X Crimson Taq
(Mg-free) Reaction Buffer
5 µl 10 μl 1X
25 mM MgCl2 Solution 1.5 μl 3 μl 1.5 mM
10 mM dNTP 0.5 µl 1 μl 200 µM
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
Crimson Taq DNA Polymerase 0.125 µl 0.25 µl 1.25 units/50 µl PCR
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 from ice to a PCR machine with the block preheated to 95°C 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. The final concentration of each primer in a 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 Crimson Taq DNA Polymerase. The Mg-free buffer formulation along with supplemental MgCl2 solution gives the user complete control over the final Mg++ concentration in the reaction.

    Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4). 

  4. Deoxynucleotides: 

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

  5. Crimson Taq DNA Polymerase Concentration: 

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

  6. Denaturation:

    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 initial 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.

  7. Annealing: 

    The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.  The NEB Tm Calculator is recommended for calculation of an appropriate annealing temperature.

    When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible (see #10).

  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 Crimson Taq 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.
3. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
4. Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res.. 18, 7465.