Haynes Lab:Notebook/CRISPR Editing/2015/02/02

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02/02/2015

PCR of gRNA from Luc14 and GAL4-EED+dox samples, g034

Cell type treatment rep Concentration ul for 200ng ul for 800ng
Luc14g034171.7742.7911.15
Luc14g0342117.5131.706.81
Luc14g0343108.0091.857.41
GAL4-EED +doxg034186.0132.339.30
GAL4-EED +doxg0342105.1271.907.61
GAL4-EED +doxg0343122.481.636.53


Mastermix 1

' 1rxn 25.5
H2030.8785.4
HF 5X MasterMix10255
dNTPs0.25.1
FP2.563.75
RP2.563.75
Total461173


Mastermix 2

' 1rxn 4.2rxns
MM146193.2
Template28.4
DMSO1.56.3
Phusion0.52.1
Total50205

Transfer 50μL of MM2 into each of 4-PCR tubes.

PCR setup

PCR

98°C for 3 min
30 cycles
98°C for 10s
69.5°C for 30s
72°C for 60s
72°C for 10 min
4°C forever


Purified with Sigma genelute PCR clean up kit. Yields aren't as high as I'd like but I can work with it, especially if it reduces my background.

Cell type treatment rep 260 280 260/280 ng/µL
Luc14g03410.0290.0171.7529.049
Luc14g03420.0290.0161.83928.848
Luc14g03430.0390.0211.83539.051
GAL4-EED +doxg03410.0390.0211.84138.915
GAL4-EED +doxg03420.0380.0192.02837.778
GAL4-EED +doxg03430.0420.0221.91442.041


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