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
Luc14 g034 1 71.774 2.79 11.15
Luc14 g034 2 117.513 1.70 6.81
Luc14 g034 3 108.009 1.85 7.41
GAL4-EED +dox g034 1 86.013 2.33 9.30
GAL4-EED +dox g034 2 105.127 1.90 7.61
GAL4-EED +dox g034 3 122.48 1.63 6.53


Mastermix 1

' 1rxn 25.5
H20 30.8 785.4
HF 5X MasterMix 10 255
dNTPs 0.2 5.1
FP 2.5 63.75
RP 2.5 63.75
Total 46 1173


Mastermix 2

' 1rxn 4.2rxns
MM1 46 193.2
Template 2 8.4
DMSO 1.5 6.3
Phusion 0.5 2.1
Total 50 205

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
Luc14 g034 1 0.029 0.017 1.75 29.049
Luc14 g034 2 0.029 0.016 1.839 28.848
Luc14 g034 3 0.039 0.021 1.835 39.051
GAL4-EED +dox g034 1 0.039 0.021 1.841 38.915
GAL4-EED +dox g034 2 0.038 0.019 2.028 37.778
GAL4-EED +dox g034 3 0.042 0.022 1.914 42.041