Haynes Lab:Notebook/CRISPR Editing/2015/03/11

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03/11/2015

PCR of g031 treated samples with phusion with primers P197/198
Include negative control

Mastermix 1 ' '
1rxn 6.5rxns
H20 30 195
HF 5X MasterMix 10 65
dNTPs 1 6.5
FP 2.5 16.25
RP 2.5 16.25
DMSO 1.5 9.75
47.5 308.75
1rxn
MM1 47.5
Template 2
Phusion 0.5
50
Cell type gRNA Rep ul for 200ng
Luc14 g025 1 0.99
Luc14 g025 2 0.97
Luc14 g025 3 0.93
Luc14 g031 1 1.15
Luc14 g031 2 0.82
Luc14 g031 3 1.08
Luc14 g032 1 1.31
Luc14 g032 2 1.13
Luc14 g032 3 1.57
Luc14 g044 1 1.07
Luc14 g044 2 1.05
Luc14 g044 3 1.44
Gal4-EED+dox g025 1 1.14
Gal4-EED+dox g025 2 0.97
Gal4-EED+dox g025 3 1.30
Gal4-EED+dox g031 1 1.21
Gal4-EED+dox g031 2 1.21
Gal4-EED+dox g031 3 1.46
Gal4-EED+dox g032 1 0.99
Gal4-EED+dox g032 2 0.92
Gal4-EED+dox g032 3 1.00
Gal4-EED+dox g044 1 1.02
Gal4-EED+dox g044 2 0.87
Gal4-EED+dox g044 3 0.90



Make 1:1000 dilutions of P197/198 PCR products and used the Luc14 and Gal4 unt dilutions to PCR again with phusion and P151/158.
Purified PCR products and ran on a gel. Single bands, nothing in any of the negative control wells.

Cell type treatment rep 260 280 260/280 ng/µL
Luc14 Unt 1 0.102 0.058 1.773 102.043
Luc14 Unt 2 0.074 0.039 1.89 73.938
Luc14 Unt 3 0.096 0.051 1.9 96.085
Gal4-EED +dox Unt 1 0.109 0.057 1.903 109.078
Gal4-EED +dox Unt 2 0.105 0.054 1.93 104.82
Gal4-EED +dox Unt 3 0.078 0.042 1.849 77.617
Luc14 g031 1 0.088 0.048 1.832 87.725
Luc14 g031 2 0.078 0.043 1.832 78.231
Luc14 g031 3 0.09 0.048 1.887 89.871
Gal4-EED +dox g031 1 0.075 0.041 1.828 74.832
Gal4-EED +dox g031 2 0.07 0.037 1.883 70.303
Gal4-EED +dox g031 3 0.084 0.046 1.805 83.843



Miniprep KAH228, cut with E and P, run on gel, send for sequencing

Check on cells

qPCR of g034 in pX330 mixed with gDNA from Gal4-EED+dox untreated rep 1. 1ng of gDNA and dilutions of plasmid. Also did gDNA dilutions and water only controls.