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Protocol/17
From 2010.igem.org
(Difference between revisions)
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|- | |- | ||
|TOTAL || 50ul | |TOTAL || 50ul | ||
| + | |} | ||
| + | * Program to run: | ||
| + | {| | ||
| + | |1. 94<sup>o</sup>C || 3 minutes | ||
| + | |- | ||
| + | |2. 94<sup>o</sup>C || 45 seconds | ||
| + | |- | ||
| + | |3. 62<sup>o</sup>C || 30 seconds | ||
| + | |- | ||
| + | |4. 72<sup>o</sup>C || 90 seconds | ||
| + | |- | ||
| + | |5. Cycle steps 1 to 4 "30" times | ||
| + | |- | ||
| + | |6. 72<sup>o</sup>C || 10 minutes | ||
|} | |} | ||
Revision as of 02:23, 26 October 2010
Protocol 17: 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 |
