General Taq PCR Master Mix -- CHEM 384/584
Ken Christensen
Abstract
SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent 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. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.
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 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 | 98C | 1 minute |
| Denature | 94C | 10 seconds |
| Anneal | 55C | 30 seconds |
| Extension | 72C | 1 min/kb |
| Repeat steps 2-4 | 30-40x | |
| Final extension | 72 | 1 minute |
| 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.
