03/10/11
- ✓ RNA prep/ cDNA: KAH126 -/+ dox
- ✓ Luc activity assay: luc repressed 6-day, Pc-TF 4-day
RNA prep
> Use TriZOL
> Dissolve each pellet in 15 μL THE RNA Storage Sln.
| Sample |
A260 |
260/280 |
ng/ul |
uL = 2ug RNA |
dH2O
|
| 1. KAH126-1, no dox |
4.284 |
1.88 |
171.3 |
do not use* |
---
|
| 2. KAH126-1, no dox |
8.057 |
1.98 |
322.3 |
6.2 |
1.8
|
| 3. KAH126-1, no dox |
16.255 |
2.01 |
650.2 |
3.1 |
4.9
|
| 4. KAH126-1, 4-day |
9.217 |
1.96 |
368.7 |
5.4 |
2.6
|
| 5. KAH126-1, 4-day |
6.483 |
2.01 |
259.3 |
7.7 |
0.3
|
| 6. KAH126-1, 4-day |
21.578 |
1.93 |
863.1 |
2.3 |
5.7
|
Note: *Accidentally added phenol:chloroform:isoamyl alcohol to cells (sample #1)
> oligo(dT) Primer annealing
--> Set up four reactions for each condition:
- Dox-: use sample #3
- Dox+: use sample #6
| Reagent |
Vol
|
| total RNA (up to 2μg) |
up to 8 μL
|
| 50μM oligo(dT) primer |
1.0
|
| 10 mM dNTP mix |
1.0
|
| DEPC-treated water |
---
|
| |
10.0 μL --> 65°C/ 5 min.; ice/ 1 min.
|
> cDNA synthesis mix
--> 16 reactions total
| Reagent |
Vol |
Mix (x8)
|
| 10x RT buffer |
2.0 |
16.0
|
| 25 mM MgCl2 |
4.0 |
32.0
|
| 0.1 M DDT |
2.0 |
16.0
|
| RNaseOUT |
1.0 |
8.0
|
| SuperScript III RT |
1.0 |
8.0
|
| |
10.0 μL |
80.0 μL
|
--> Add 10 μL mix to each annealing rxn.
--> 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 0.8 μL RNase H, 37°C/ 20 min.
--> Store at -20°C
Luc Activity Assay
Luc activity assay
> Cells: dox-induced Gal4-EED expression/ 2 days, followed by transfection for 4 days
1-6. KAH160/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid
7-12. KAH170/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid
> Sample processing:
- Add 500 μL medium to cells in rep 1 set (measured before); Resuspend cells in 1 mL medium total
- Aliquot 3x 100 μL samples into 96-well plate
- Save the rest for cell count (Millipore Scepter)
- Normalize each value to cell count
|
Project name
