Team:UCSF/Protocols

From 2010.igem.org

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'''3) Vortex and spin down the reaction.'''<br>
'''3) Vortex and spin down the reaction.'''<br>
'''4) Set up PCR program.'''<br>
'''4) Set up PCR program.'''<br>
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'''General PCR program'''
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''Note: Times may differ for different reactions based on the size of the desired PCR product. Annealing temperature may also differ depending on primer properties.''
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Revision as of 11:22, 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.

General PCR program

Cycle StepTemperatureTime# Cycles
Initial Denature98C3min1
Denature
Annealing
Extension
98C
55C
72C
10s
30s
30s
30
Final Extension72C5min1
Hold4C

Note: Times may differ for different reactions based on the size of the desired PCR product. Annealing temperature may also differ depending on primer properties.