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 0.25 0.8
g034 0.5 1.6
g034 0.75 2.4
g034 1.0 3.2


Step 2: make DNA + optimem mixes

' 1 well 3.2 x
g034 0.25 39 add 124.8ul optimem
g034 0.5 39 add 124.8ul optimem
g034 0.75 39 add 124.8ul optimem
g034 1.0 39 add 124.8ul optimem


Step 3: add plus reagent to DNA+ opti

' 1 well 3.2 x
g034 0.25 1 add 3.2ul plus reagent
g034 0.5 1 add 3.2ul plus reagent
g034 0.75 1 add 3.2ul plus reagent
g034 1.0 1 add 3.2ul plus reagent


Step 4: Make optimem + lipo mastermix

' 1 well for 12 wells (x 13.2)
optimem 47 620.4
lipo 3 39.6


Step 5: mix dna mix and lipo mix

g034 0.25 add 160 lipo mix
g034 0.5 add 160 lipo mix
g034 0.75 add 160 lipo mix
g034 1.0 add 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