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Team:University College London/Protocols/Week12/3
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Revision as of 22:02, 26 September 2012 by Erinoerton (Talk | contribs)
IrrE PCR
| Gene to be amplified | Primers |
|---|---|
| irrE | Forward primer: STF1: 5'-atggggccaaaagctaaagctgaagcc-3' Reverse primer: ST2R: 5'-tcactgtgcagcgtcctgcg-3' |
| irrE | Forward primer: STF3 5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3' Reverse primer: ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3' |
All primers were prepared to a concentration of 1pmol.uL, from 100pmol.uL stocks. Primers ordered from MWG operon.
| Component | 20 µl reaction | 50 µl reaction | Final concentration |
|---|---|---|---|
| Nuclease-free water | to 20 µl | to 50 µl | |
| 5X Phusion HF buffer | 4 µl | 10 µl | 1X |
| 10 mM dNTPs | 0.4 µl | 1 µl | 200 µM |
| 10 µM forward primer | 1 µl | 2.5 µl | 0.5 µM |
| 10 µM reverse primer | 1 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 250 ng |
| DMSO (optional) | (0.6 µl) | (1.5 µl) | 3% |
| Phusion DNA polymerase | 0.2 µl | 0.5 µl | 1.0 units/50 µl PCR |
