Haynes Lab:Notebook/CRISPR Editing/2015/07/14

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07/14/2015

collected siRNA transfected cells from one well each (dox vs puro).

washed with PBS
trypsin
collect with media into 1.5mL tube
spin at 0.2g for 3 mins at bench
remove media
wash with 1mL cold PBS
spin at 0.2g for 3 mins at bench
remove PBS
resuspend with 500uL cold PBS


luciferase assay, in triplicate, 100ul cells, 100ul luciferin
mix with pipette then orbital shaker for 10 mins (usually only do 5)
count cells in 10ul volume

Results

Gal4-EED+dox cells seem to be reopened, about 1AU/cell vs 0-0.2 that I usually see.
Gal4-EED+puro cells may have been opened even more, about 3AU/cell vs 1 that I usually see
Could be that I left too long, considering redoing it tomorrow, i have another extra well
will proceed with transfections because it seems like the siRNAs did reopen the Gal4+dox cells to some extent



Transfecting siRNA treated cells with g034 and g048
Will transfect gal4-eed+dox and gal4-eed+puro with each gRNA in triplicate
Step 1: Diluting the DNA

' ug for 1 well ug of DNA for 6.2 wells ul plasmid water (62ul total)
g034 (118ng/ul) 0.5 3.1 26.27 35.73
g048 (222ng/ul) 0.5 3.1 13.96 48.04

Step 2: make DNA + optimem mixes
add 241.8ul optimem to each tube

' ul opti for 1 well 3.2 x
g034 39 241.8
g048 39 241.8

Step 3: add plus reagent to DNA+ opti
add 6.2ul plus reagent to each tube

' 1 well 6.2 wells
g034 1 6.2
g048 1 6.2

Step 4: Make optimem + lipo mastermix
Mix lipo and optimem. let sit for 5 minutes

' 1 well for 12 wells (x 12.8)
optimem 47 592.2
lipo 3 37.8

Step 5: mix dna mix and lipo mix
add 310 lipo-opti mix to each tube. let sit for 30 mins

' 1 well 6.2 wells
g034 50 310
g048 50 310