05/30/11
- ✓ Transfection: miR sensor constructs into U2OS plain
Lipo LTX transfection
> Follow Jason's protocol, 12-well plates
> Set up each in triplicate, 48 samples (4 plates) total
> Keep 5th plate as "blank" control for FACS
Wells |
Plasmid |
DNA |
Volume |
PLUS reagent |
Lipo |
Opti-MEM (total)
|
1-3 |
FC1* |
1 μg |
### μL |
1 uL |
3 μL |
200 μL
|
4-6 |
FC2 |
" |
### μL |
" |
" |
"
|
7-9 |
FC3 |
" |
### μL |
" |
" |
"
|
10-12 |
FC4 |
" |
### μL |
" |
" |
"
|
13-15 |
FC31 |
" |
### μL |
" |
" |
"
|
16-18 |
FC32 |
" |
### μL |
" |
" |
"
|
19-21 |
FC33 |
" |
### μL |
" |
" |
"
|
22-24 |
FC34 |
" |
### μL |
" |
" |
"
|
25-27 |
KAH100 |
" |
### μL |
" |
" |
"
|
28-30 |
KAH101 |
" |
### μL |
" |
" |
"
|
31-33 |
KAH118* |
" |
### μL |
" |
" |
"
|
34-36 |
KAH119* |
" |
### μL |
" |
" |
"
|
37-39 |
KAH121 |
" |
### μL |
" |
" |
"
|
40-42 |
KAH122 |
" |
### μL |
" |
" |
"
|
43-45 |
KAH124 |
" |
### μL |
" |
" |
"
|
46-48 |
KAH125 |
" |
### μL |
" |
" |
"
|
- Poor quality DNA (see BioBrick Cloning entry 5/30/11)
> Dilute 1 ug DNA in 20 μL (total) st dH2O; set up 3x and aliquot 20 μL to other 2 tubes
> Add 180 μL Opti-MEM
> Add 1 μL PLUS reagent --> R.T/ 5 min.
> Add 3 μL Lipo LTX --> R.T/ 30 min.
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C
|