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Team:Washington/GENERAL PCR PROTOCOL
From 2013.igem.org
Untitled Document General PCR Protocol
See also: PCR_GoTaq
Resuspending primers:
Protocol: Resuspending PCR primers
Working Stock:
(serves as a backup)
Dilute to 10 times the number on primer vial
10ul fwd primer 90ul DI water
10ul rev primer 90ul DI water
Notes for iGEM 2013 HSB lab:
We are using the GreenTaq polymerase 2X pre-mixed (GoTaq) for PCR.
Use the protocols below for Green Taq polymerase.
Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).
If PCRing insert use 1ng/ul DNA concentration.
Amplification PCR with Green Taq polymerase:
Setup Amplification PCR Reaction
1uL Template DNA
22ul deionized (dI) H2O
1ul 10uM forward primer (Tm XX C)*
1ul 10uM reverse Primer (Tm YY C)*
25ul 2x Green taq (GoTaq)
Total 50ul
Amplification PCR Reaction Program
95 C for 30s
95 C for 30s
ZZ* C for 30s (important step)
72 C for 1min per kb target gene plus 10-15% (important step)
Repeat 2-4 34x
72 C for 5 to 10min
4 C for forever (don’t forget to turn the machine off the next day)
Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).
Analytical PCR with Green Taq polymerase:
Setup PCR Reaction
1uL Template
2ul dI H2O
1ul 10uM forward primer (Tm XX C)*
1ul 10uM reverse Primer (Tm YY C)*
5ul 2x Green taq (GoTaq)
Total 10uls
PCR Reaction Program
95 C - 30s
95 C - 10s
ZZ* C - 10s (important step)
72 C - 1min per kb target gene plus 10-15% (important step)
Repeat b-d 25x
72 C - 5 to 10min
10 or 4 C - forever
Phusion polymerase:
Setup Amplification PCR Reaction
1uL Template
1uL 25mM dNTP's
10uL Phusion HF Buffer
1.0ul Forward Primer (Tm XX oC)*
1.0ul Reverse Primer (Tm YY oC)*
0.5uL Phusion polymerase
36.5uL diH2O
Total 50uls
Amplification PCR Reaction Program
98 C - 30s
98 C - 10s
ZZ* C - 10s (important step)
72 C - 30s per kb target gene plus 10-15% (important step)
Repeat b-d 29-34x
72 C - 5 to 10min
10 or 4 C - forever
Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).
Refer for further details to NEB's online protocol for Phusion:
Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase
