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

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

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/┬ÁL
pMax GFP 6.23501.1190.6051.851119.431
g048 in pX330g1280.2030.1091.862203.431
g034 in pX330g1180.1840.0971.905184.145
pX330g1840.3940.2061.909394.082



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


Personal tools