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)
|
0.32 |
|
|
g034 0.01 |
0.032 |
|
|
g034 0.1 |
0.32 |
|
|
g034 0.01 |
0.032 |
|
|
Step 2: make DNA + optimem mixes
'
|
1 well
|
3.2 x
|
g034 0.1 |
39 |
add 124.8ul optimem
|
g034 0.01 |
39 |
add 124.8ul optimem
|
g034 0.1 |
39 |
add 124.8ul optimem
|
g034 0.1 |
39 |
add 124.8ul optimem
|
Step 3: add plus reagent to DNA+ opti
'
|
1 well
|
3.2 x
|
g034 0.1 |
1 |
add 3.2ul plus reagent
|
g034 0.01 |
1 |
add 3.2ul plus reagent
|
g034 0.1 |
1 |
add 3.2ul plus reagent
|
g034 0.01 |
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 0.1 |
add 160 lipo mix
|
g034 0.01 |
add 160 lipo mix
|
g034 0.1 |
add 160 lipo mix
|
g034 0.01 |
add 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
|