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
|
| 
Project name
Main project page
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