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-EED Puro g032 1 0.102 0.052 1.953 102.256 3.91 1.50 9.59
Gal4-EED Puro g032 2 0.093 0.05 1.873 93.185 4.29 1.50 9.21
Gal4-EED Puro g032 3 0.069 0.036 1.907 69.418 5.76 1.50 7.74
Gal4-EED Dox g032 1 0.083 0.043 1.941 83.246 4.81 1.50 8.69
Gal4-EED Dox g032 2 0.087 0.045 1.912 86.929 4.60 1.50 8.90
Gal4-EED Dox g032 3 0.092 0.049 1.902 92.435 4.33 1.50 9.17
Gal4-EED Puro g046 1 0.081 0.042 1.913 80.615 4.96 1.50 8.54
Gal4-EED Puro g046 2 0.076 0.041 1.834 75.671 5.29 1.50 8.21
Gal4-EED Puro g046 3 0.077 0.04 1.927 76.588 5.22 1.50 8.28
Gal4-EED Dox g046 1 0.063 0.034 1.864 63.022 6.35 1.50 7.15
Gal4-EED Dox g046 2 0.081 0.042 1.909 80.568 4.96 1.50 8.54
Gal4-EED Dox g046 3 0.071 0.035 2 70.905 5.64 1.50 7.86

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-EED dox/puro EZH2 g025 1 0.18 0.087 2.069 179.613
Gal4-EED dox/puro EZH2 g025 2 0.173 0.087 2.004 173.433
Gal4-EED dox/puro EZH2 g025 3 0.104 0.051 2.054 104.127
Gal4-EED dox/puro Blank g025 1 0.201 0.099 2.033 201.109
Gal4-EED dox/puro Blank g025 2 0.234 0.115 2.034 233.53
Gal4-EED dox/puro Blank g025 3 0.174 0.086 2.027 174.093
Gal4-EED dox/puro EZH2 g034 1 0.097 0.048 2.024 96.926
Gal4-EED dox/puro EZH2 g034 2 0.062 0.032 1.939 62.154
Gal4-EED dox/puro EZH2 g034 3 0.124 0.061 2.015 123.599
Gal4-EED dox/puro Blank g034 1 0.214 0.108 1.989 214.213
Gal4-EED dox/puro Blank g034 2 0.213 0.106 2.018 213.397
Gal4-EED dox/puro Blank g034 3 0.182 0.09 2.034 182.355