12/17/09
- ✓ Transfection: RFP reporter into JDS37-43 (luc miRNA-YFP)
Transfections
> U2OS JDS37-43 (luc miRNA-YFP) cells + RFP-luc target reporters
> Use Lipofectamine, 6-well format
| Wells |
Plasmid |
DNA |
Volume |
Lipo |
Opti-MEM (total)
|
| 1 |
KAH93/MV2 |
2 μg |
4.4 μL |
4 μL |
500 μL
|
| 2 |
KAH94/MV2 |
" |
4.8 μL |
" |
"
|
| 3 |
KAH95/MV2 |
" |
8.7 μL |
" |
"
|
| 4 |
KAH10/pcN |
" |
4.5 μL |
" |
"
|
| 5 |
no DNA |
--- |
--- |
" |
"
|
| 6 |
untreated |
--- |
--- |
--- |
"
|
Day 1 (today)
> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Remove 0.5 mL growth medium from each well before adding transf. mix. Add 500 μL complexes to each well (total vol./well = 4 mL); Grow cells at 37°C
> After 5 hours, change medium (ab-free)
Day 2 (12/18/09)
> Transfection (DNA+Lipo) was slightly toxic, no need to split cells; change medium to 15μg/mL puro(morning)
> Do microscopy to find RFP+ cells (record positions using Metamorph multi-position staging)
> Add 1 μg/mL doxycycline
Day 3
> Microscopy: record RFP vs. YFP expression at set stage positions
Day 4
> Repeat Day 3
| 
Project name
Main project page
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