Haynes Lab:Notebook/CRISPR Editing/2014/09/21

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Ran the PCR for gRNA 20 samples from yesterday on a gel. Ran 3μL of each sample and 2μL and 1μL of ladder. Only the first three worked, even though the other two worked the other day.

Annealing all of each of gRNA 20, plus the PCRs from the other day that worked. Will just go through the protocol to see if I can even do Surveyor with the concentrations I'm getting.

Going to try more PCR. Will do gRNA 21 samples and all the Luc14 samples. If I can get all the Luc14 samples through the entire protocol, I can know if any of the gRNAs aren't cutting at all and won't have to keep trying the chromatin-induced samples.

Set 1 Set 2
gRNA 21 + dox 1 gRNA 20 Luc 14
gRNA 21 + dox 2 gRNA 21 Luc 14
gRNA 21 - dox 1 gRNA 22 Luc 14
gRNA 21 - dox 2 gRNA 27 Luc 14
gRNA 21 Luc 14 gRNA 29 Luc 14

Master mix 1

' 1rxn 10.5 rxns
H20 23.5 246.75
5x buffer 10 105
dNTPs 1 10.5
FP 2.5 26.25
RP 2.5 26.25
DMSO 1.5 15.75

add 41ul of MM 1 to each tube
add 4ul of each template to each tube (400ng each) Master mix 2

' 1rxn 10.5 rxns
H20 4.5 47.25
Phusion 0.5 5.25

add 5 ul of MM 2 to each tube

98°C for 3 min
36 cycles
98°C for 10s
66°C for 30s
72°C for 60s
72°C for 10 min
4°C forever

transferred 40ul of the first three annealing reactions and 35ul of the last two to new tubes. added 1ul enhancer and 2ul of surveyor nuclease. incubate at 42deg C for 1 hour.
added 5ul of water to whatever is left in the PCR tubes from annealing. will run those as a no-nuclease control.

Ran Surveyor of gRNA 20 samples on 1% gel

Ran PCR products of gRNA 21 samples and all Luc 14 samples on gel. Annealed.