Team:UCSF/Protocols

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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 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.

Ligation Protocol

Ligation Reagents:
50 ng vector
DNA insert(s)
Buffer
1 ul Ligase

Ligation Reaction:
1. Calculate amount of DNA insert and vector needed for reaction.
150 ng / ( # bp in backbone/ # bp in insert) = ng needed
Concentration of insert or vector divided by ng needed = volume of DNA needed
2. Calculate amount of buffer needed depending on concentration for volume needed. A typical ligation reaction volume can be 20 ul.
3. Add reagents together, from smallest volumes to largest. Distilled water can be used to bring the volume up so that the reaction has the proper buffer concentration for the reaction. Ligase or enzymes in general should be added at the end.
4. Vortex or pipet up and down to mix. Spin down afterwards if vortexing.
5. Let reaction sit at room temperature for 5 to 20 minutes depending on ligase used. The ligase may start acting up if left too long.