02/08/10
- ✓ RNA preps/ cDNA synth.: Polycomb-ATF lines (dox-induced, ~2 days)
- ✓ HEK293 #4 Luc: 1x 1:10 flask, 1x 24-well plate (~10,000 cells per well) for Gal4-EED transfection/ luc assay
- ✓ U20S Flp-in T-REx: 1x 1:10 flask, 1x 6-well (for Pc-ATF gene expression control)
RNA Preps/ cDNA synth.
> RNA Preps
--> Use 1 mL TriZol/ well (6-well dish)
--> Combine duplicate after resuspending RNA pellet in 6 μL THE RNA Storage Solution
| Sample |
OD 260 |
260/280 |
ng/μL |
vol = 2 μg
|
| 1. KAH126-1 |
10.051 |
2.01 |
402.1 |
4.5 μL
|
| 2. KAH127-4 |
6.877 |
1.87 |
275.1 |
7.5 μL
|
| 3. KAH128-8 |
52.594 |
1.72 |
676.3 |
3.0 μL
|
| 4. KAH129-4 |
7.789 |
1.94 |
311.6 |
6.5 μL
|
| 5. KAH130-2 |
8.398 |
1.95 |
335.9 |
6.0 μL
|
| 6. KAH131-9 |
6.813 |
1.81 |
173.0 |
8.0 μL
|
| 7. KAH132-8 |
51.697 |
1.76 |
923.0 |
2.0 μL
|
| 8. KAH133-1 |
5.709 |
1.84 |
228.4 |
8.0 μL
|
> oligo(dT) Primer annealing
| Reagent |
Vol
|
| total RNA (2 μg) |
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
--> 8 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 1.0 μL RNase H, 37°C/ 20 min.
--> Store at -20°C
|
Project name
Main project page
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