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Team:Alberta/Notebook/protocols/pcr
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Revision as of 01:34, 27 October 2010
PCR
Procedure:
- Before you start:
- KEEP EVERYTHING ON ICE. Put Taq back into freezer as soon as you`re done with it. DON`T put back dNTP tubes.
- Reserve a thermocycler and check what size of tubes it takes.
- Making a master mix conserves expensive reagents, so try to always use one.
- You may have to do dilutions of your reagents in order to make them usable for PCR (ex: primers, plasmid DNA...)
- DON'T PUT PRIMERS OR TEMPLATE INTO THE MASTER MIX. ADD POLYMERASE TO THE MASTER MIX LAST (after your other tubes already have template DNA and primers in them)!
- To add into a tube:
| PCR buffer | 5ul |
| 10uM dNTPs | 1ul |
| 50uM MgCl2 | 2ul |
| Forward primer | 2.5ul |
| Reverse primer | 2.5ul |
| 1ng Template | 1ul |
| Taq polymerase | 0.5ul |
| MilliQ water | 35.5ul |
| TOTAL | 50ul |
- Program to run:
| 1. 94oC | 3 minutes |
| 2. 94oC | 45 seconds |
| 3. 62oC | 30 seconds |
| 4. 72oC | 90 seconds |
| 5. Cycle steps 1 to 4 "30" times | |
| 6. 72oC | 10 minutes |
