IGEM:Paris Bettencourt 2012/Protocols/PCR Jake
Extraction of Genomic DNA
1) Pick a single colony into 50 ul of H20.
Fresh colonies (grown that day) work best, but they can also come from 4 C.
2) Boil for 5 minutes.
1 ul of this can be used directly for PCR.
Best if used directly, but can also be stored at 4 C for a few days.
PCR Recipe (NEB Phusion Polymerase)
We use the Phusion polymerase because it has a low mutation rate. It also has a very fast extension time, which is useful for amplifying large constructs.
|Thermocycler Protocol: NEB Phusion|
|Start||98 C||30 sec||Melt|
|Cycle 1||98 C||5 sec||Melt|
|Cycle 2||55 C||25 sec||Anneal|
|Cycle 3||72 C||30 sec per kb||Extend|
|Finish||72 C||5 min||Extend|