Haynes Lab:Notebook/CRISPR Editing/2015/10/02

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10/02/2015

Spec'd and set up annealing reaction for g032 and g046 +/- chromatin

Cell type Drug gRNA rep 260 280 260/280 ng/µL ul for 400ng ul ann buff ul H20
Gal4-EEDPurog03210.1020.0521.953102.2563.911.509.59
Gal4-EEDPurog03220.0930.051.87393.1854.291.509.21
Gal4-EEDPurog03230.0690.0361.90769.4185.761.507.74
Gal4-EEDDoxg03210.0830.0431.94183.2464.811.508.69
Gal4-EEDDoxg03220.0870.0451.91286.9294.601.508.90
Gal4-EEDDoxg03230.0920.0491.90292.4354.331.509.17
Gal4-EEDPurog04610.0810.0421.91380.6154.961.508.54
Gal4-EEDPurog04620.0760.0411.83475.6715.291.508.21
Gal4-EEDPurog04630.0770.041.92776.5885.221.508.28
Gal4-EEDDoxg04610.0630.0341.86463.0226.351.507.15
Gal4-EEDDoxg04620.0810.0421.90980.5684.961.508.54
Gal4-EEDDoxg04630.0710.035270.9055.641.507.86

Set up g032 for surveyor, 1ul surveyor, 1ul enhancer, 60mins at 42degC, add stop solution, dilute 1:20, run on BA

Ran g054, g055 nested PCR on gel. g054 looked good, a faint band in the negative control lane. g055 was faint, no band in negative control lane.

Isolated gDNA from siRNA samples. EZH2 and blank transfection, then either g025 or g034, in triplicate.

Cell type drug siRNA gRNA rep 260 280 260/280 ng/µL
Gal4-EEDdox/puroEZH2g02510.180.0872.069179.613
Gal4-EEDdox/puroEZH2g02520.1730.0872.004173.433
Gal4-EEDdox/puroEZH2g02530.1040.0512.054104.127
Gal4-EEDdox/puroBlankg02510.2010.0992.033201.109
Gal4-EEDdox/puroBlankg02520.2340.1152.034233.53
Gal4-EEDdox/puroBlankg02530.1740.0862.027174.093
Gal4-EEDdox/puroEZH2g03410.0970.0482.02496.926
Gal4-EEDdox/puroEZH2g03420.0620.0321.93962.154
Gal4-EEDdox/puroEZH2g03430.1240.0612.015123.599
Gal4-EEDdox/puroBlankg03410.2140.1081.989214.213
Gal4-EEDdox/puroBlankg03420.2130.1062.018213.397
Gal4-EEDdox/puroBlankg03430.1820.092.034182.355



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