The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
PcTF Subcloning in E. coli
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
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
PlpE = 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 (3:1) + FlpE |
" |
4.42 + 2.60 μL |
12.98μL |
" |
" |
"
|
1-3 |
BD002 (4:1) + FlpE |
" |
4.73 + 2.60 μL |
12.67μL |
" |
" |
"
|
1-4 |
BD002 (4:1)+ FlpE |
" |
4.73 + 2.60 μL |
12.67μL |
" |
" |
"
|
1-5 |
BD002 (3:1) + --- |
" |
4.42 + --- |
15.58μL |
" |
" |
"
|
1-6 |
BD002 (4:1) + --- |
" |
4.73 + --- |
15.58μL |
" |
" |
"
|
- 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.
|