Haynes Lab:Notebook/CRISPR Editing/2015/12/01

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12/01/2015

left four plasmids with lids open in the TC hood overnight with the sash closed

plasmid orig conc 260 280 260/280 ng/µL
pMax GFP 6.2 350 1.119 0.605 1.85 1119.431
g048 in pX330g 128 0.203 0.109 1.862 203.431
g034 in pX330g 118 0.184 0.097 1.905 184.145
pX330g 184 0.394 0.206 1.909 394.082



Transfection with pMax GFP 6.2 into Luc14 Cell density (cells/mL): 5 x 107
Transfection efficiency: 80%
Viability: 80%
Tip type: 100ul

prewarm four 10cm plates with 10mLs antibiotic-free DMEM

put plasmid DNA into 1.5mL tube :5ug = 4.47ul (10ul other) :10ug = 8.93ul (20ul other) :20ug = 17.87ul (40ul other)

add 3mL E2 buffer to a neon tube
set up device :Pulse voltage (v): 1,100 :Pulse width (ms): 20 :Pulse number: 2

collect all the cells :wash and spin and wash and spin :resuspend in 450mL Resuspension buffer R add cells to DNA
pull cells+DNA into gold tip
start
eject cells into plate, rock plate, incubate