General Taq PCR Master Mix -- CHEM 384/584
Ken Christensen
Abstract
2X PCR Master Mixes are convenient to use as they include all the necessary PCR components except the template and primers. Most PCR Master Mixes also include agarose gel running dyes and a density reagent that allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR.
Steps
Setup Reaction
To a 25µL aliquot of a 2X Taq PCR Master Mix (e.g. TaqDog, or Sapphire Amp), add template (10-20 µl cleared lysate for colony PCR or 20-50ng of purified DNA for typical PCR), forward and reverse primers to a final concentration of 200nanomolar (nM). Adjust final volume to 50µL with nuclease free water or autoclaved water.
| A | B |
|---|---|
| 2X Master Mix | 25 ul (pre-aliquoted and stored in the freezer) |
| Template | 10-20 ul of bacterial lysate or 20-50 ng purified DNA |
| Forward Primer | 1 ul of 10 uM primer dilution |
| Reverse Primer | 1 ul of 10 uM primer dilution |
| ddH2O | to a final volume of 50 ul |
Run Reaction
followed by 30 cycles of 98°C, 5 sec; 55°C, 5 sec; and 72°C, 40 sec.
| A | B | C |
|---|---|---|
| Initial denature | 94C | 1 minute |
| Denature | 94C | 30 seconds |
| Anneal | 55C | 30 seconds |
| Extension | 72C | 1 min/kb |
| Repeat steps 2-4 | 25-40x | |
| Final extension | 72 | minutes |
| Cool | 4C | Until cancelled |
A typical thermocycling program for a PCR for amplicons less than 1 kb is 1 minute. For longer amplicons, adjust the program to 1 minute/kb seconds for the extension and final extension times.
