PCR Protocol for OneTaq® DNA Polymerase (M0480)
New England Biolabs
Abstract
The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification. Taq DNA Polymerase is an enzyme widely used in PCR. One Taq DNA Polymerase allows for greater amplification sensitivity across a wide variety of amplicons regardless of GC content. The following guidelines are provided to ensure successful PCR using New England Biolabs’ One Taq DNA Polymerase. These guidelines cover most routine PCR. Specialized applications may require further optimization.
Before start
Steps
Set up the following reaction On ice:
| A | B | C | D |
|---|---|---|---|
| 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 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 |
Gently mix the reaction.
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.
Quickly transfer PCR tubes to a thermocycler preheated to the denaturation temperature (94°C) and begin thermocycling.
Perform PCR using the general routine below or using your own optimized routine:
| A | B | C |
|---|---|---|
| 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/kb |
| Final Extension | 68°C | 5 minutes |
| Hold | 4-10°C |
