02/20/10
- ✓ RNA/ cDNA prep: 4-day dox Pc-ATF cultures (plate set A)
- ✓ Pc-ATF RT-PCR cultures: split plate set B into two duplicates; start new plate for KAH130-2
Cytology: seed 24-well cover slip plate for H3K27me/ H3K9me staining Very sparse cell growth. Do over on Tuesday
- ✓ Cultures: Split confluent Pc-ATF lines 1:10 (10 cm plates)
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 |
2.347 |
1.98 |
93.9 |
8.0 μL (0.75 μg)
|
2. KAH126-1 dox |
1.295 |
1.94 |
51.8 |
8.0 μL (0.41 μg)
|
3. KAH127-4 |
2.722 |
2.01 |
108.9 |
8.0 μL (0.87 μg)
|
4. KAH127-4 dox |
0.723 |
2.17 |
28.9 |
8.0 μL (0.23 μg)
|
5. KAH128-8 |
11.440 |
1.91 |
457.6 |
4.4 μL (2.00 μg)
|
6. KAH128-8 dox |
9.511 |
1.94 |
380.4 |
5.2 μL (2.00 μg)
|
7. KAH129-4 |
24.166 |
2.01 |
966.6 |
2.1 μL (2.00 μg)
|
8. KAH129-4 dox |
0.885 |
2.00 |
35.4 |
8.0 μL (0.28 μg)
|
9. KAH130-2 |
1.617 |
2.01 |
64.7 |
8.0 μL (0.52 μg)
|
10. KAH130-2 dox |
83.459 |
1.61 |
3338.4 |
0.6 μL (2.00 μg)
|
11. KAH131-9 |
33.028 |
2.00 |
1321.1 |
1.5 μL (2.00 μg)
|
12. KAH131-9 dox |
0.328 |
2.31 |
13.1 |
8.0 μL (0.11 μg)
|
13. KAH132-8 |
2.941 |
1.70 |
117.6 |
8.0 μL (0.94 μg)
|
14. KAH132-8 dox |
4.776 |
1.98 |
191.0 |
8.0 μL (1.53 μg)
|
15. KAH133-1 |
15.005 |
1.97 |
600.2 |
3.3 μL (2.0 μg)
|
16. KAH133-1 dox |
37.503 |
2.00 |
1500.1 |
1.3 μL (2.0 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
--> 8 reactions total
Reagent |
Vol |
Mix (x16)
|
10x RT buffer |
2.0 |
32.0
|
25 mM MgCl2 |
4.0 |
64.0
|
0.1 M DDT |
2.0 |
32.0
|
RNaseOUT |
1.0 |
16.0
|
SuperScript III RT |
1.0 |
16.0
|
|
10.0 μL |
160.0 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
|