Haynes Lab:Notebook/CRISPR Editing/2014/09/23

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09/23/2014

Running 2ul of each PCR rxn on a gel, with 3ul of water and 1ul of 6x loading dye.
figuring out volumes of wells so I can know exactly what volume to use for each annealing reaction.
purifying and concentrating all successful PCR rxns using column. should also figure out if 50x loading dye works.

Sample Read# 260 280 260/280 ng/µL ul for 400ng
Luc14 gRNA 20 0.141 0.075 1.864 140.539 2.85
Luc14 gRNA 21 0.176 0.098 1.788 175.643 2.28
Luc14 gRNA 22 0.198 0.106 1.869 197.974 2.02
Luc14 gRNA 27 0.131 0.07 1.868 131.014 3.05
Luc14 gRNA 29 0.159 0.086 1.858 159.485 2.51

Set up annealing reaction, 10ul total

' ul for 400ng ul elution solution ul water ul 10x annealing buffer
gRNA 20 2.85 0.15 6 1
gRNA 21 2.28 0.72 6 1
gRNA 22 2.02 0.98 6 1
gRNA 27 3.05 -0.05 6 1
gRNA 29 2.51 0.49 6 1


Added 1ul of enhancer and 1ul of nuclease to each reaction, incubate at 42 for 1 hour.
Ran gel 2ul of ladder, 1ul of each purified PCR reaction (lost a little of gRNA 22), and gRNA 27 unpurified reaction 2 that looked like it didn't work at all.
uncut controls of each gRNA purified pcr of the same volumes as annealing reactions.