IGEM:Paris Bettencourt 2012/Protocols/PCR Jake

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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.


Reagent
Volume
1x
Nuclease-free water
37 ul
5x Phusion HF Buffer
10 ul
10 mM dNTPs
1 ul
Forward Primer (10 uM)
0.5 ul
Reverse Primer (10 uM)
0.5 ul
Genomic DNA
1 ul
Phusion DNA Polymerase
0.5 ul ul
Total Volume
50 ul


Thermocycler Protocol: NEB Phusion
Temp Time
Start 98 C 30 sec Melt
Cycle 1 98 C 5 sec Melt
35 Cycles
Cycle 2 55 C 25 sec Anneal
Cycle 3 72 C 30 sec per kb Extend
Finish 72 C 5 min Extend
Store 10 C Forever Store
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