Team:UCSF/Protocols
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Revision as of 11:20, 18 October 2010
AarI Digest Protocol
1) Label PCR tubes.
2) Add the following reagents into the PCR tubes:
Aar1 Digest Reagents
5 ug DNA
2.5ul Aar1 Enzyme
0.9ul Aarl oligo
6 ul 10x Aar1 Buffer
x ul dH2O
60 ul total reaction
Note: x ul dH2O may change in volume for different reactions in order to bring total volume up to 60 ul.
3) Briefly vortex and spin down the reaction.
4) Incubate reaction at 37C for 3 hours.
Note: PCR program can be set to 37C for 3 hours and cooled down to 4C indefinitely.
PCR Phusion Protocol
1) Label PCR tubes.
2) Add the following reagents into the PCR tubes (in order):
PCR Reagents
23.7 ul dH2O
10 ul 5x HF Buffer
5 ul forward primer
5 ul reverse primer
5 ul dNTP
0.3 ul template
1 ul Phusion
50 ul total
3) Vortex and spin down the reaction.
4) Set up PCR program.
Cycle Step | Temperature | Time | # Cycles | Initial Denature | 98C | 3min | 1 |
Denature Annealing Extension | 98C 55C 72C | 10s 30s 30s | 30 | ||||
Final Extension | 72C | 5min | 1 | ||||
Hold | 4C |