06/06/2013
Minipreps
> Check with E, P, and E/P digests
Reagent |
1,4,7,10 |
2,5,8,11 |
3,6,9,12
|
Expected: 1. FlpE, E = 7160, 546 2. FlpE, P = 7706 3.FlpE, E/P=5649, 1511, 546
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0 |
2.0 |
2.0
|
10X buffer |
1.5 |
1.5 |
1.5
|
EcoRI |
1.0 |
--- |
1.0
|
PstI |
--- |
1.0 |
1.0
|
dH2O |
10.5 |
10.5 |
9.5
|
|
|
15 μL --> 37°C/ ~15 min.
--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)
--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.
- ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
- ✓ Polycomb-ATF negative control lines: colony plates
1 day before transfection:
- Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.
Transfections
> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:
- BD002 (3:1) Flp-in T-REx
- BD002 (4:1) Flp-in T-REx
BD002 (3:1) = 339 ng/μl
BD002 (4:1) = 317 ng/μl
KAH187 = 441.5 ng/μl
FlpE = 192 ng/μl
Plate-Well |
Plasmid |
DNA |
Volume |
dH2O |
Lipo |
PLUS reagent |
Opti-MEM (total)
|
1-1 |
BD002 (3:1) + FlpE |
1.5 + 0.5 μg |
4.42 + 2.60 μL |
12.98μL |
2.5μL |
7.5 μL |
570 μL
|
1-2 |
BD002 (4:1) + FlpE |
" |
4.73 + 2.60 μL |
12.67μL |
" |
" |
"
|
1-3 (-) Ctrl |
BD002 (3:1) + --- |
2 μg |
4.42 + --- μL |
15.58μL |
" |
" |
"
|
1-4 (-)Ctrl |
--- + --- |
0 μg |
--- + --- μL |
20.0μL |
" |
" |
"
|
1-5 |
KAH187 |
20 μg |
4.53 |
15.46μL |
" |
" |
"
|
- Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
- Label sterile microfuge (1.5 ml) tubes.
- Add 570 μL Opti-MEM to each 20 μL DNA sample.
- Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
- Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
- Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
- Incubate cells at 37°C in a CO2 incubator
- (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
Transgene expression should be detectable after 18 hours.
|