Haynes Lab:Notebook/CRISPR Editing/2015/09/14

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09/14/2015

nested pcr of g025 with P159/215, 1:1000 dilution. Ran on gel, no bands in -ctrl lane. purified PCR, set up annealing rxn for surveyor

Cell type drug gRNA rep 260 280 260/280 ng/µL ul for 400ng ul ann buff ul water
Gal4-EED puro g025 1 0.06 0.031 1.946 60.017 6.66 1.50 6.84
Gal4-EED puro g025 2 0.053 0.028 1.863 53.041 7.54 1.50 5.96
Gal4-EED puro g025 3 0.03 0.016 1.869 29.68 13.48 1.50 0.02
Gal4-EED dox g025 1 0.054 0.031 1.73 54.062 7.40 1.50 6.10
Gal4-EED dox g025 2 0.056 0.03 1.868 55.579 7.20 1.50 6.30
Gal4-EED dox g025 3 0.054 0.03 1.787 53.892 7.42 1.50 6.08

nested pcr of g044 with P213/214, 1:1000 dilution. Ran on gel, no bands in -ctrl lane. purified PCR, set up annealing rxn for surveyor

Cell type drug gRNA rep 260 280 260/280 ng/µL ul for 400ng ul ann buff ul water
Gal4-EED puro g044 1 0.064 0.034 1.858 63.523 6.30 1.50 7.20
Gal4-EED puro g044 2 0.059 0.032 1.856 58.653 6.82 1.50 6.68
Gal4-EED puro g044 3 0.054 0.03 1.816 53.743 7.44 1.50 6.06
Gal4-EED dox g044 1 0.061 0.032 1.871 60.721 6.59 1.50 6.91
Gal4-EED dox g044 2 0.061 0.033 1.836 60.606 6.60 1.50 6.90
Gal4-EED dox g044 3 0.055 0.029 1.914 54.571 7.33 1.50 6.17

collected siRNA transfected, g025 or 34 transfected cells (washed, trypsin, collect, spin 3min at 0.2RCF, remove sup, resuspend in 300uL PBS), run 100ul on flow, started gDNA extraction on the remaining 200ul (20ul proteinase k, 200ul buffer al, vortex 15s, 10min at 57deg, put at -20).