12/31/09
- ✓ RNA prep/ cDNA synth.: KAH93, 94, 95/MV2 transfected cells (+/- Dox)
RNA prep & cDNA
> 6-well plates: use 1 mL TRIZol per well
> Dissolve pellet in 10 μL THE RNA Storage Solution
> Measure RNA concentrations
| Sample |
A260 |
260/280 |
ng/μL |
vol = 2 μg
|
| 1. KAH93/MV2 |
8.866 |
1.98 |
354.7 |
5.6 μL
|
| 2. KAH93/MV2 (Dox) |
10.554 |
1.96 |
422.2 |
4.7
|
| 3. KAH94/MV2 |
16.045 |
1.90 |
641.8 |
3.1
|
| 4. KAH94/MV2 (Dox) |
15.813 |
1.90 |
632.5 |
3.2
|
| 5. KAH95/MV2 |
15.973 |
1.95 |
638.9 |
3.1
|
| 6. KAH95/MV2 (Dox) |
41.982 |
1.97 |
1679.3 |
1.2
|
> oligo(dT) Primer annealing
--> details
| Reagent |
Vol
|
| total RNA (up to 5μg) |
2 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
--> 6 reactions total
| Reagent |
Vol |
Mix (x12)
|
| 10x RT buffer |
2.0 |
12.0
|
| 25 mM MgCl2 |
4.0 |
24.0
|
| 0.1 M DDT |
2.0 |
12.0
|
| RNaseOUT |
1.0 |
6.0
|
| SuperScript III RT |
1.0 |
6.0
|
| |
10.0 μL |
60.0 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
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