Team:Alberta/Notebook/protocols/pcr

From 2010.igem.org

Revision as of 17:11, 26 October 2010 by Wiltshir (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

TEAM ALBERTA

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


Back

Retrieved from "http://2010.igem.org/Team:Alberta/Notebook/protocols/pcr"