PCR Reactions
Gregory W. Broussard
Revised: 8/3/07
(Please use, change and disseminate freely!)
- Use primers at a concentration of 10 μM
- Using DMSO at 5% may help the reaction, especially with DNA of high GC content (ex: Mycobacterium chromosomal DNA). For M. smegmatis and mycobacteriophages, I now always use 5% DMSO.
- 50 μl total volume
- This protocol is for Pfu DNA Polymerase (Stratagene)
- For mycobacteriophages as template, use 1μl of lysate in place of template DNA.
- PCR Rxn Mix:
| Components | Stock Concentration | Final reaction Concentration |
50 ml reactiona |
| M. smegmatis DNA | 5 – 25 ng/μl | 0.1 – 0.5 ng/μl |
1 μl |
| Primer A | 10 μM | 0.25 μM |
1.25 μl |
| Primer B | 10 μM | 0.25 μM |
1.25 μl |
| dNTP mixture | 10 mM | 0.2 mM |
1 μl |
| 10X buffer | 10X | 1X |
5 μl |
| DMSOb | 100% | 5% |
2.5 μl OR 0 μl |
| Pfu | 2.5 U/μl | 2.5 U |
1 μl |
| dH2O | — | — |
37 μl OR 39 μl |
- Annealing temperature should be determined by looking at the melting temperature (Tm) for both primers. It is recommended to choose an annealing temperature 2 °C lower that the lowest Tm.
- Extension time for small PCR products (< 1000 bp) should be 1 min. 30 sec.
- PCR:
| Cycle function | Number of cycles | Temperature | Time length |
| Initial denaturation | 1 | 95°C | 5 min |
| Denaturation | 95°C | 30 sec | |
| Annealinga | 30 | —— | 30 sec |
| Extension | 72°C | 1 min/kb + 30 sec | |
| Final Extension | 1 | 72°C | 7 min |
| Hold | 1 | 4°C | Indefinite |
- Run 5 μl of the PCR product on a gel.
Leave a Comment
Gregory W. Broussard, Ph.D. 