TITLE: PCR Protocol for Taq DNA Polymerase with ThermoPol® Buffer (M0267)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
PCR Protocol for Taq DNA Polymerase with ThermoPol® Buffer (M0267).

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

[GUIDELINES]
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 NEB's 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.

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:
DNAAmountgenomic1 ng–1 μgplasmid or viral1 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 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 Taq DNA Polymerase. The final Mg++ concentration in 1X ThermoPol Reaction Buffer is 2 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 (3) or formamide (4).

4. Deoxynucleotides: 

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

5. Taq DNA Polymerase Concentration: 

We generally recommend using Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of 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 Denaturation95°C30 seconds30 Cycles95°C

11. PCR product: 

The PCR products generated using 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.

[STEPS]
1. Assemble the following reaction on ice:
   Component 25 μl reaction 50 μl reaction Final Concentration   10X ThermoPol Reaction Buffer 2.5 µl 5 μl 1X   10 mM dNTPs 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   Taq DNA Polymerase 0.125 µl 0.25 µl 1.25 units/50 µl PCR   Nuclease-free water to 25 µl to 50 µl     

 

2. Gently mix the reaction.
3. Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid.
4. Quickly transfer PCR tubes from ice to a PCR machine with the block preheated to 95 °C and begin thermocycling. 
6. Perform PCR using the general routine below or using your own optimized routine: 
 

5. Select between thermocycling conditions for a routine PCR and for a routine 2-step PCR:
 

6. Thermocycling conditions for a routine 2-step PCR: