Making up 10X PCR buffer (Promega recipe, Mg free )
This solution must be made up as cleanly as possible to prevent contamination in everyone's PCR reactions. The following is one attempt to do that.
1.Clean pipettors
Soak white barrels in 1 N HCl for 1/2 hour. Rinse with water and then distilled water. Dry in drying oven. If in a hurry, you can rinse with ethanol to get rid of any water and then dry in the oven.
2. Tips
Use barrier tips and 25 ml sterile pipettes for pipetting. All solutions should be autoclaved prior to mixing (except for Triton X).
3. Make up in 50 ml purple capped, sterile tubes.
I usually make two batches (2 tubes or 40 ml). Then I aliquot one and freeze the other.
4. Recipe
| Solution | Volume to add | 10X buffer concentration | Working PCR concentration |
| 1 M KCl | 20 ml | 500 mM | 50 mM |
| 1 M Tris HCl pH 9.0 | 4 ml | 100 mM | 10 mM |
| Triton-X | 0.4 ml | 1% | 0.1% |
| Sterile distilled water | 15.6 ml | ||
| Total | 40 ml |
5. Checking
Try PCR on known primers including negative controls. Make sure there is no amplification in negatives, but is amplification in positives.
6. Aliquotting
Use O ring tubes. Aliquot exactly 1.0 ml per tube using barrier tips.