Haynes Lab:Notebook/CRISPR Editing/2016/05/24

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05/24/2015

Added RNase to g025 sonicated chromatin, 30min at 37

finally got enough DNA from PCR for g025 3. Means there's probably something wrong with my Phusion.

Cell type gRNA rep 260 280 260/280 ng/µL ul for 400ng ul hyb buff ul H2O
Luc14 g025 3 0.034 0.019 1.809 33.885 11.80 1.50 1.70

Can run surveyor on g025 and 31 today. Set up annealing reaction:

Cell type gRNA rep 260 280 260/280 ng/µL ul for 400ng ul hyb buff ul water
Luc14 25 1 0.035 0.02 1.792 35.279 11.34 1.50 2.16
Luc14 25 2 0.046 0.025 1.876 46.254 8.65 1.50 4.85
Luc14 25 3 0.034 0.019 1.809 33.885 11.80 1.50 1.70
Luc14 31 1 0.07 0.037 1.9 69.982 5.72 1.50 7.78
Luc14 31 2 0.087 0.046 1.885 86.915 4.60 1.50 8.90
Luc14 31 3 0.035 0.019 1.868 35.484 11.27 1.50 2.23



Added proteinase k to 25 sonicated chromatin. 62 for 2 hours

conc of sheared chromatin

Cells gRNA rep 260 280 260/280 ng/µL ul for 50ug ul dil buff
Luc14 25 1 2.272 1.32 1.721 2271.864 22.01 177.99
Luc14 25 2 2.426 1.42 1.708 2426.165 20.61 179.39
Luc14 25 3 2.022 1.17 1.728 2021.664 24.73 175.27
Gal4 25 1 2.128 1.267 1.68 2127.688 23.50 176.50
Gal4 25 2 1.305 0.795 1.643 1305.148 38.31 161.69
Gal4 25 3 1.903 1.138 1.672 1902.941 26.28 173.72