Team:UCSF/Protocols

(Difference between revisions)

Revision as of 11:17, 18 October 2010

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.

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

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