06/02/13
- Transfection: KAH126 for flow cytometry on Monday
- Gal4EED/luc test: induce repression with dox
Lipo transfection
> Cells were 100% confluent
> Re-plate 1:2 dilution prior to transfection in 3.4 mL p/s-free medium
Wells |
Plasmid |
DNA |
Volume + dH2O |
Opti-MEM |
PLUS |
Lipo |
|
1 |
KAH126 |
2 μg |
9.0 μL +11.0 |
570 μL |
2.5 μL |
7.5 μL
|
2 |
KAH126 |
1 μg |
4.5 μL +15.5 |
" |
" |
"
|
3 |
KAH126 |
0.5 μg |
2.3 μL +17.7 |
" |
" |
"
|
4 |
KAH126 |
0.2 μg |
0.9 μL +19.1 |
" |
" |
"
|
5 |
KAH126 |
0.1 μg |
0.5 μL +19.5 |
" |
" |
"
|
6 |
none |
none |
--- |
none |
none |
none
|
> Add 570 μL Opti-MEM to each 20 μL DNA sample
> Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. 5 min/room temp
> Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. 30 min/ room temp
> Add 600 μL complexes to each well (final volume of medium = 4 ml/well)
> Add 1 μg/μL dox
> Grow cells at 37°C overnight
Next day
> Harvest cells for flow cytometry at Biodesign (contact Tong Xu)
Gal4EED/luc test
> Dox dose response test
> 6-well plate (plated 5/29/13), shipment 1 cells
Wells |
dox μg/mL |
Volume dox (1 mg/mL) in 3.5 mL medium
|
1 |
0 |
---
|
2 |
0.1 |
0.4 μL
|
3 |
0.2 |
0.8 μL
|
4 |
0.5 |
1.8 μL
|
5 |
1.0 |
3.5 μL
|
6 |
2.0 |
7.0 μL
|
|