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- 92 - 94oC for 30 - 60 sec (denature)
- 37 - 72oC for 30 - 60 sec (anneal)
- 72oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length)
- 25 - 35 cycles only (otherwise enzyme decay causes artifacts)
- 72oC for 5 min at end to allow complete elongation of all product DNA
NOTE:
"Quickie" PCR is quite feasible: eg, [94oC 30 sec / 45oC 30 sec / 72oC 30 sec] x 30, for short products (200 - 500 bp).
YOU CAN USE GLYCEROL IN THERMAL CYCLER REACTION TUBE HOLES TO ENSURE GOOD THERMAL CONTACTS
DON'T RUN TOO MANY CYCLES: if you don't see a band with 30 cycles you probably won't after 40; rather take an aliquot from the reaction mix and re-PCR with fresh reagents. See here.
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