Haynes Lab:Notebook/CRISPR Editing/2015/03/22

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03/22/2015

Column purified unt and g032 treated nested PCR with P149/174. Will set up hybridization of 400ng PCR product in 15ul reaction:

Cell type treatment rep 260 280 260/280 ng/µL ul for 400ng ul water ul hyb buff total
Luc14 Unt 1 0.036 0.02 1.805 36.09 11.08 2.42 1.5 15
Luc14 Unt 2 0.042 0.024 1.801 42.349 9.45 4.05 1.5 15
Luc14 Unt 3 0.038 0.021 1.815 38.219 10.47 3.03 1.5 15
Gal4-EED+dox Unt 1 0.045 0.025 1.777 45.01 8.89 4.61 1.5 15
Gal4-EED+dox Unt 2 0.042 0.023 1.836 42.28 9.46 4.04 1.5 15
Gal4-EED+dox Unt 3 0.038 0.022 1.77 38.201 10.47 3.03 1.5 15
Luc14 g032 1 0.035 0.018 1.982 35.035 11.42 2.08 1.5 15
Luc14 g032 2 0.04 0.022 1.787 39.676 10.08 3.42 1.5 15
Luc14 g032 3 0.042 0.024 1.785 42.176 9.48 4.02 1.5 15
Gal4-EED+dox g032 1 0.043 0.023 1.851 42.765 9.35 4.15 1.5 15
Gal4-EED+dox g032 2 0.037 0.022 1.65 36.658 10.91 2.59 1.5 15
Gal4-EED+dox g032 3 0.038 0.022 1.742 37.775 10.59 2.91 1.5 15


Annealing reaction in Surveyor protocol
1ul surveyor enzyme, 1ul enhancer, 60 mins at 42deg
Dilute 1:10 in ES, run on bioanalyzer