12/24/09
- ✓ RNA prep & cDNA for KAH93, 94, 95, 108/MV2 transfected Dox+/- JDS33-47 cells
- ✓ Split U2OS plain: 1x 1:10 flask; 2x 6-well, 2.5x105/well, ab-free
- ✓ Split JDS33-47: 1x 1:10 flask
RNA prep & cDNA
> 6-well plates: use 1 mL TRIZol per well
> Dissolve pellet in 15 μL THE RNA Storage Solution
> Measure RNA concentrations
| Sample |
A260 |
260/280 |
ng/μL |
vol = 2 μg
|
| 1. KAH93/MV2 |
7.657 |
1.98 |
306.9 |
6.5
|
| 2. KAH93/MV2 (Dox) |
0.060 |
6.73 |
2.4 |
x
|
| 3. KAH94/MV2 |
10.346 |
1.97 |
413.8 |
4.8
|
| 4. KAH94/MV2 (Dox) |
0.517 |
1.82 |
20.7 |
x
|
| 5. KAH95/MV2 |
18.231 |
1.88 |
729.3 |
2.7
|
| 6. KAH95/MV2 (Dox) |
15.123 |
1.92 |
604.9 |
3.3
|
| 7. KAH108/MV2 |
26.328 |
1.98 |
1053.1 |
1.9
|
| 8. KAH108/MV2 (Dox) |
23.386 |
2.00 |
935.4 |
2.1
|
| 9. no DNA |
23.601 |
2.01 |
944.1 |
2.1
|
| 10. no DNA (Dox) |
17.490 |
2.00 |
699.6 |
2.9
|
> oligo(dT) Primer annealing
--> Discard low yeild samples (2, 4); retry later
--> 2 rxns for KAH108, 108/Dox, no DNA, no DNA/ Dox (12 total)
| Reagent |
Vol
|
| total RNA |
up to 5 μg
|
| 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
| Reagent |
Vol |
Mix (x12)
|
| 10x RT buffer |
2.0 |
24.0
|
| 25 mM MgCl2 |
4.0 |
48.0
|
| 0.1 M DDT |
2.0 |
24.0
|
| RNaseOUT |
1.0 |
12.0
|
| SuperScript III RT |
1.0 |
12.0
|
| |
10.0 μL |
120.0 μL
|
--> Add 10 μL mix to each annealing rxn.
--> 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 1.0 μL RNase H, 37°C/ 20 min.
--> Store at -20°C
|
Project name
Main project page
Previous entry Next entry
