EpiMark® Hot Start Taq DNA Polymerase Guidelines for PCR (M0490)
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. EpiMark® Hot Start Taq DNA Polymerase allows for greater PCR sensitivity and permits room temperature PCR reaction set-up. The following guidelines are provided to ensure successful PCR using New England Biolabs’ EpiMark Hot Start Taq DNA Polymerase. These guidelines cover routine PCR.
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.
Protocol
Reaction Setup:
Add to a sterile thin-walled PCR tube:
| Component | 25 µl Reaction |
50 µl Reaction |
Final Concentration |
| 5X EpiMark® Hot Start Taq Reaction Buffer |
5 µl | 10 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 | 0.5µl | 1 µl | 0.2 µM (0.05-1 µM) |
| EpiMark® 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 cycler and begin thermocycling:
Thermocycling conditions for a routine PCR:
Initial denaturation:
95°C 30 seconds
35-40 cycles:
95°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
General Guidelines:
- Template:
Use of high quality, bisulfite-treated DNA templates greatly enhances the success of reactions. 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
- Primers:
Oligonucleotide primers for bisulfite-converted DNA are generally 25–40 nucleotides in length, and should not contain CpG sites within their sequence to ensure unbiased amplification of both methylated or unmethylated DNA. Primer design and analysis can be facilitated using programs such as MethPrimer or BiSearch. methBLAST allows you to check your primers or bisulfite-converted DNA by blasting them against the unmethylated and methylated genomic sequences of human, mouse and rat. The recommended Tm for a primer set can be determined using the New England Biolabs Tm calculator. The final concentration of each primer in a reaction may be 0.05–1 µM, typically 0.2 µM.
- Mg++ and additives:
Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with EpiMark Hot Start Taq DNA Polymerase. The final Mg++ concentration in 1X EpiMark Hot Start Taq Reaction Buffer is 1.8 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2 (NEB# B9021).
- Deoxynucleotides:
The final concentration of dNTPs is typically 200 µM of each deoxynucleotide.
- EpiMark Hot Start Taq DNA Polymerase Concentration:
We generally recommend using EpiMark Hot Start Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 µl reaction). However, the optimal concentration of EpiMark Hot Start Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 µl reaction) in specialized applications.
- Denaturation:
No separate activation step is required to release the hot start inhibitor from the enzyme. An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can further damage bisulfite-converted DNA and is not recommended.
During thermocycling a 15–30 second denaturation at 95°C is recommended.
- Annealing:
The annealing step is typically 15–60 seconds. The 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.
- 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.
- Cycle number:
Generally, 35–40 cycles yield sufficient product. Up to 45 cycles may be required to detect low copy number targets from bisulfite-converted DNA.
- PCR product:
The PCR products generated using EpiMark Hot Start Taq DNA Polymerase contain dA overhangs at the 3´ end; therefore the PCR products can be ligated to dT/dU-overhang vectors.