Protocol for OneTaq Hot Start DNA Polymerase (M0481)
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. OneTaq Hot Start DNA Polymerase allows for greater amplification sensitivity across a wide variety of amplicons and increased ease of reaction setup. The following guidelines are provided to ensure successful PCR using New England Biolabs' OneTaq Hot Start DNA Polymerase. These guidelines cover most routine PCR. Specialized applications may require further optimization.
Protocol
Reaction setup:
Due to the presence of the inhibitor, 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 |
|---|---|---|---|
| 5X OneTaq Standard Reaction Buffer* |
5 µl | 10 μl | 1X |
| 10 mM dNTPs (#N0447) | 0.5 µl | 1 μl | 200 µM |
| 10 µM Forward Primer | 0.5 µl | 1 μl | 0.2 µM |
| 10 µM Reverse Primer | 0.5 µl | 1 μl | 0.2 µM |
| OneTaq Hot Start 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 |
*OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons.
**For amplicons between 3–6 kb, use 2.5–5 units/50 µl rxn
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 |
94°C |
30 seconds |
| 30 Cycles | 94°C 45-68°C 68°C |
15-30 seconds 15-60 seconds 1 minute per kb |
| Final Extension | 68°C |
10 minutes |
| Hold | 4-10°C |
General Guidelines:
- 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
- Primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as PrimerSelect™ (DNAStar Inc., Madison, WI) and Primer 3 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.
- Mg++ and Additives:
Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with OneTaq Hot Start DNA Polymerase. The final Mg++ concentration in 1X OneTaq Standard Reaction 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
- Deoxynucleotides:
The final concentration of dNTPs is typically 200 µM of each deoxynucleotide.
- OneTaq Hot Start DNA Polymerase Concentration:
We generally recommend using OneTaq Hot Start DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 µl reaction) for amplicons up to 3 kb. The optimal concentration of OneTaq Hot Start DNA Polymerase may range from 5–100 units/ml (0.25–5 units/50 µl reaction) for specialized applications. For 3–6 kb amplicons, 2.5–5 units/50 µl reaction is recommended. Note that in some cases, increasing the amount of enzyme in the reaction can be inhibitory.
- Denaturation:
No separate activation step is required to release the hot start inhibitor from the enzyme. An initial denaturation of 30 seconds at 94°C is sufficient to amplify most targets from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2–4 minutes at 94°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 2–5 minute incubation at 94°C is recommended to lyse cells.
During thermocycling a 10–30 second denaturation at 94°C is recommended.
- 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.
- 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, 25–35 cycles yield sufficient product. Up to 45 cycles may be required to detect low copy number targets.
- 2-step PCR:
When primers with annealing temperatures of 68°C or above are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
- PCR Product:
A significant portion of the PCR products generated using OneTaq Hot Start DNA Polymerase contain dA overhangs at the 3´end; therefore the PCR products can be ligated to dT/dU-overhang vectors.