06/15/10
- ✓ Western: protein prep 126-1, 126-3, 132-8 -/+ dox, 4 days (from 6-well)
- ✓ RT-PCR (set #1): 126-1, 126-3, 132-8, 154-2, FTRx -/+ dox, 4 days RNA preps (from 6-well); cDNA
- ✓ Cell culture: split cells 1:10 (10 cm plates)
- ✓ RT-PCR (set #2): start new 6-well cultures (-/+ dox) for 126-1, 126-3, 132-8, 154-2, FTRx
RNA Preps/ cDNA synth.
> RNA Preps
--> Use 1 mL TriZol/ well (6-well dishes)
--> Resuspend RNA pellet in 10 μL THE RNA Storage Solution
| Sample |
OD 260 |
260/280 |
ng/μL |
Vol. to use for cDNA synth.
|
| 1. KAH126-1 |
41.982 |
1.99 |
1679.3 |
3.0 μL (5 μg)
|
| 2. KAH126-1 dox |
27.348 |
2.00 |
1093.9 |
4.5 μL (5 μg)
|
| 3. KAH126-3 |
51.548 |
1.98 |
2061.9 |
2.5 μL (5 μg)
|
| 4. KAH126-3 dox |
22.083 |
1.93 |
883.3 |
6.0 μL (5 μg)
|
| 5. KAH132-8 |
55.233 |
1.96 |
2209.3 |
2.5 μL (5 μg)
|
| 6. KAH132-8 dox |
11.019 |
1.93 |
440.8 |
8.0 μL (3.5 μg)
|
| 7. KAH154-2 |
42.558 |
1.98 |
1702.3 |
3.0 μL (5 μg)
|
| 8. KAH154-2 dox |
37.529 |
1.98 |
1501.2 |
3.5 μL (5 μg)
|
| 9. FTRx |
55.342 |
1.97 |
2213.7 |
2.5 μL (0.52 μg)
|
| 10. FTRx dox |
39.820 |
1.92 |
1592.8 |
3.0 μL (2.00 μg)
|
> oligo(dT) Primer annealing
| 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
--> 10 reactions total
| Reagent |
Vol |
Mix (x10)
|
| 10x RT buffer |
2.0 |
20.0
|
| 25 mM MgCl2 |
4.0 |
40.0
|
| 0.1 M DDT |
2.0 |
20.0
|
| RNaseOUT |
1.0 |
10.0
|
| SuperScript III RT |
1.0 |
10.0
|
| |
10.0 μL |
100.0 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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