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PCR screening
The following protocol is established for the screening of 10 colonies.
Preparation of the PCR mixture (final volume 200 µl)
Component Volume Final concentration
| Component | Volume | Final concentration |
| H2O | 150 µl | |
| 10 x PCR buffer | 20 µl | |
| 20 x dNTP | 10 µl | each nucleoside 250 µM |
| downstream primer* | 10 µl | 400 µM |
| upstream primer* | 10 µl | 400 µM |
| Taq polymerase | 3µl | 1 U |
- divide the PCR mixture in 10 aliquots (20 µl each);
- with a sterilised toothpick, slightly touch the surface of the colony in order to pick up a very small amount of material;
- dip the end of the toothpick into the PCR mixture for 2-3 seconds and then remove it;
- add two drops of mineral oil;
-
the conditions on the termal cycler are the following:
94 °C (10 min) - [94 °C (1 min) - 55 °C (1 min) - 72 °C (2 min)]30 - 65 °C (2 min)
For phagemids, the two primers are LMB3 and FdSeq
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