Haynes Lab:Notebook/CRISPR Editing/2015/06/27

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06/27/2015

transfected gal4-eeds with puro and dox with four dilutions of g034 in triplicate. did the following twice, once for each drug treatment

Step 1: Diluting the DNA

' ug of DNA for 3.2 wells ul plasmid water (32ul total)
g034 0.250.8
g034 0.51.6
g034 0.752.4
g034 1.03.2


Step 2: make DNA + optimem mixes

' 1 well 3.2 x
g034 0.2539add 124.8ul optimem
g034 0.539add 124.8ul optimem
g034 0.7539add 124.8ul optimem
g034 1.039add 124.8ul optimem


Step 3: add plus reagent to DNA+ opti

' 1 well 3.2 x
g034 0.251add 3.2ul plus reagent
g034 0.51add 3.2ul plus reagent
g034 0.751add 3.2ul plus reagent
g034 1.01add 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 0.25add 160 lipo mix
g034 0.5add 160 lipo mix
g034 0.75add 160 lipo mix
g034 1.0add 160 lipo mix

Step 6: add 100ul to each well



Collected transfected luc14 cells 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



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