08/25/2015
Transfect gal4+puro and gal4+dox with g034, collect at 24, 48, and 72 hours
Step 1: Making .5ug/10ul plasmid stocks
| '
|
1 well
|
19 wells
|
| ug g034 |
0.5 |
9.50
|
| ul g034 |
|
|
| ul water |
|
|
Step 2: make DNA + optimem mixes
| '
|
1 well
|
19 x
|
| g034 |
39 |
add 741ul optimem
|
Step 3: add plus reagent to DNA+ opti
| '
|
1 well
|
| g034 |
add 19ul plus reagent
|
step 4: Make optimem + lipo mastermix
| '
|
1 well
|
for 18 wells (x 19.2)
|
| optimem |
47 |
902.4
|
| lipo |
3 |
57.6
|
Step 5: mix dna mix and lipo mix
| '
|
1 well
|
x 19
|
| g034 |
50 |
add 950 lipo mix
|
Step 6: add lipo mix to cells
add 100ul of mix to each well
Transfected siRNAs EZH2 and SUZ12 into gal4-EEDs with dox
Cells plated yesterday looked good.
- Cells plated at 30% were at about 80%
- Cells plated at 10% and 20% were at about 50%
Transfected 2 wells for each siRNA and each cell type
For siRNA complexes
| '
|
per well
|
3.1
|
| 20uM siRNA duplex |
2.5 |
7.75
|
| Optimem |
46 |
142.6
|
| Oligofectamine |
1.5 |
4.65
|
| Total |
50 |
155
|
For blank transfections
| '
|
per well
|
3.1
|
| water |
2.5 |
7.75
|
| Optimem |
46 |
142.6
|
| Oligofectamine |
1.5 |
4.65
|
| Total |
50 |
155
|
Mix optimem and siRNA or water
- 7.75ul each siRNA + 124ul Optimem in 1.5mL tube
- 7.75ul water + 124ul Optimem in 2 1.5mL tubes
Mix oligofectamine and optimem
- 19.8ul oligofectamine + 79.2ul optimem
- let stand for 10 minutes
Mix DNA and oligofectamine
- add 23.25ul oligofectamine to tubes with RNA or water
- let stand for 30 minutes
While complexes form, prep cells
- Removed media
- Wash in 100ul PBS
- Aspirate PBS
- Add 200ul of plain DMEM
Add complexes to cells
- Add 50ul complexes dropwise to each well
After 4 hours, add 125ul DMEM + 3x serum to each well.
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Project name
Main project page
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