Haynes Lab:Notebook/CRISPR Editing/2015/06/30

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06/30/2015

transfected Luc14s gal4-eeds with dox with 2 dilutions of g034 in triplicate.

Step 1: Diluting the DNA

' ug of DNA for 3.2 wells ul plasmid water (32ul total)
g034 0.10.32
g034 0.010.032
g034 0.10.32
g034 0.010.032


Step 2: make DNA + optimem mixes

' 1 well 3.2 x
g034 0.139add 124.8ul optimem
g034 0.0139add 124.8ul optimem
g034 0.139add 124.8ul optimem
g034 0.139add 124.8ul optimem


Step 3: add plus reagent to DNA+ opti

' 1 well 3.2 x
g034 0.11add 3.2ul plus reagent
g034 0.011add 3.2ul plus reagent
g034 0.11add 3.2ul plus reagent
g034 0.011add 3.2ul plus reagent


Step 4: Make optimem + lipo mastermix

' 1 well for 12 wells (x 13.2)
optimem47620.4
lipo339.6


Step 5: mix dna mix and lipo mix

g034 0.1add 160 lipo mix
g034 0.01add 160 lipo mix
g034 0.1add 160 lipo mix
g034 0.01add 160 lipo mix

Step 6: add 100ul to each well



Luciferase assay on Luc14s, Gal4-EEDs+puro, and Gal4-EEDs+dox (6/22 and 6/26)



Collected transfected gal4-eed cells with dox or puro for flow and gDNA extraction:

aspirate media
add 400ul PBS, rock plate, aspirate PBS
add 200ul trypsin, let sit for a few minutes, tap plate to unstick cells
add 500ul media, pipette to collect cells, move to 1.5mL microcentrifuge tube
remove tubes from hood
spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes
remove media with P1000
resuspend in 400ul cold PBS
filter 200ul for flow, use remaining 200ul for gDNA extraction




Plating cells for transfection with siRNAs with oligofectamine
oligofectamine calls for no antibiotics, serum-free media, and for cells to be 30-50% confluent at time of transfection

trypsinized Gal4-EED cells that had been induced with dox at 3PM on 6/26
filtered some DMEM only for serum-free media
Plated 0.1x10^6 cells per well of a 6-well plate



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