Haynes Lab:Notebook/CRISPR Editing/2015/05/21

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05/21/2015

Picked colonies from KAH228 in MV2 and KAH60 in pCvN transformation plates to miniprep tomorrow

Specd plasmid dilution nested PCR products

Cell type Plasmid ug plasmid ul lipo Rep 260 280 260/280 ng/µL
Gal4-EED g034 0.5 3 1 0.026 0.013 1.984 26.083
Gal4-EED g034 0.5 3 2 0.029 0.015 1.896 28.889
Gal4-EED g034 1 3 1 0.025 0.013 1.95 24.681
Gal4-EED g034 1 3 2 0.023 0.011 2.037 22.943
Gal4-EED g034 0.5 5 1 0.026 0.015 1.771 26.425
Gal4-EED g034 0.5 5 2 0.026 0.014 1.886 26.035
Gal4-EED g034 1 5 1 0.024 0.012 1.942 24.063
Gal4-EED g034 1 5 2 0.029 0.015 1.878 28.85

Annealed for surveyor, 20ul annealing reactions because of the lower concentrations

Cell type Plasmid ug plasmid ul lipo Rep ng/µL ul for 400ng ul anealing buff ul water
Gal4-EED g034 0.5 3 1 26.083 15.34 2.00 2.66
Gal4-EED g034 0.5 3 2 28.889 13.85 2.00 4.15
Gal4-EED g034 1 3 1 24.681 16.21 2.00 1.79
Gal4-EED g034 1 3 2 22.943 17.43 2.00 0.57
Gal4-EED g034 0.5 5 1 26.425 15.14 2.00 2.86
Gal4-EED g034 0.5 5 2 26.035 15.36 2.00 2.64
Gal4-EED g034 1 5 1 24.063 16.62 2.00 1.38
Gal4-EED g034 1 5 2 28.85 13.86 2.00 4.14

1ul surveyor, 1ul enhancer into tubes 1-8, 1ul water, 1ul enhancer into tube 9, 42degC for 1hour
2.2ul stop solution, ran on ba