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 ' '
1rxn6.5rxns
H2030195
HF 5X MasterMix1065
dNTPs16.5
FP2.516.25
RP2.516.25
DMSO1.59.75
47.5308.75
1rxn
MM147.5
Template2
Phusion0.5
50
Cell type gRNA Rep ul for 200ng
Luc14g02510.99
Luc14g02520.97
Luc14g02530.93
Luc14g03111.15
Luc14g03120.82
Luc14g03131.08
Luc14g03211.31
Luc14g03221.13
Luc14g03231.57
Luc14g04411.07
Luc14g04421.05
Luc14g04431.44
Gal4-EED+doxg02511.14
Gal4-EED+doxg02520.97
Gal4-EED+doxg02531.30
Gal4-EED+doxg03111.21
Gal4-EED+doxg03121.21
Gal4-EED+doxg03131.46
Gal4-EED+doxg03210.99
Gal4-EED+doxg03220.92
Gal4-EED+doxg03231.00
Gal4-EED+doxg04411.02
Gal4-EED+doxg04420.87
Gal4-EED+doxg04430.90



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/┬ÁL
Luc14Unt10.1020.0581.773102.043
Luc14Unt20.0740.0391.8973.938
Luc14Unt30.0960.0511.996.085
Gal4-EED +doxUnt10.1090.0571.903109.078
Gal4-EED +doxUnt20.1050.0541.93104.82
Gal4-EED +doxUnt30.0780.0421.84977.617
Luc14g03110.0880.0481.83287.725
Luc14g03120.0780.0431.83278.231
Luc14g03130.090.0481.88789.871
Gal4-EED +doxg03110.0750.0411.82874.832
Gal4-EED +doxg03120.070.0371.88370.303
Gal4-EED +doxg03130.0840.0461.80583.843



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.


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