Haynes Lab:Notebook/CRISPR Editing/2014/09/22

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09/22/2014

Cut the annealed Luc14 reactions, but not gRNA 21 because that one didn't amplify. added 40ul of annealed reaction to a new PCR tube, added 1ul of enhancer and 2ul of Surveyor nuclease. Incubated at 42°C for one hour, kept on ice after. Forgot to add stop solution. ran gel after about an hour or so on ice.
Ran gel:


Still unable to see the cut bands clearly. Can't conclude either way if it's working. If I get higher PCR yields, I can get 400ng in a smaller volume and run on a gel with smaller combs.



Added 100ul of elution butffer (Sigma elution solution from genelute miniprep kit) to each stock of gDNA for each sample. Then did the dilutions from before but into 20ul of elution buffer.
PCR of gRNA 20 + dox 1 (known to work consistently). Tried at lower gDNA concentrations from Bill's suggestion.
also changed the way I set up mastermixes, again from Bill's suggestion. Now make one with water, 5X buffer, and enzyme. Bill said maybe the water was affecting the enzyme.
Mastermix 1

' 1rxn 4.5 xns
H2023.5105.75
dNTPs14.5
FP2.511.25
RP2.511.25
DMSO1.56.75
31139.5

add 31ul of MM 1 to each tube
Add template to each tube

' dilution of sample ul added ul water final amount in ng
1~50ng/ul22100
2~50ng/ul0.5425
3~5ng/ul2210
4~5ng/ul0.542.5


Mastermix 2

' 1rxn 4.5 rxns
5x buffer1045
H204.520.25
Phusion0.52.25
15

add 15 ul of MM 2 to each tube
PCR

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


Ran PCR on gel

Ran dilutions of ladder on gel with tiniest comb size to determine detection threshold using Sybr and our new gel imager.


Set up 4-50ul PCR rxns for each Luc14 samples (20 total). Made one master mix of water, buffer, dNTPs, and primers, split into 5 tubes, added template, DMSO, and Phusion, then split each of those 5 into 4 tubes. Ran overnight.


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