09/14/10
- ✓ Midiprep check: KpnI digests
- ✓ ChIP/ Co-IP: wash & elute (see 9/13/10)
- ✓ RT-PCR: p16 induction cDNA from 9/13/10
Midiprep check
> Check with KpnI digests
| Reagent |
Volume
|
Expected:
Backbone = 7489
Empty vector frag. = 913
1-3. KAH160/KAH015-1,2,3 = 2060
2. KAH161/KAH015 = 2060
4-6. KAH170/KAH015-1,2,3 = 1868
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
1.0 μL
|
| 10X buffer |
1.5
|
| KpnI |
1.0
|
| dH2O |
11.5
|
| |
15 μL --> 37°C/ ~15 min.
|
|
RT-PCR
> Use cDNA from "p16 time course" samples
> Same PCR conditions as p16 (see 8/26/10)
> Templates + Primers:
- p16 time course (1-16) + p16 7C (0.5 uL cDNA)
- p16 time course (1-16) + mCherry f1/r1 (1.0 uL 1:1000 cDNA dln.)
- p16 time course (1-16) + GAPDH 21A (0.5 uL 1:1000 cDNA dln.)
| Reagent |
1-16, MM (x17) |
17-32, MM (x17) |
33-48, MM (x17)
|
| cDNA |
--- (0.5/ rxn) |
--- (1.0/ rxn) |
--- (0.5/rxn)
|
| 10 μM primers |
17.0 (p16 7C) |
17.0 (mCh f1/r1) |
17.0 (GAPDH 21A)
|
| 2x GoTaq Green |
170.0 |
170.0 |
170.0
|
| dH2O |
144.5 |
136.0 |
144.5
|
| |
20.0 μL/rxn
|
--> Aliquot MM's: 19.5 for 1-16, 19.0 for 17-32, 19.5 for 33-48
--> Add cDNA
--> PCR (96-well)*
- 95°C/ 3 min.
- [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
- 72°C/ 3 min.
- 4°C/ ∞
(*Same as p16INK PCR, 8/26/10)
> Conclusions: No signal for p16. Re-make cDNA with 5 ug RNA. Non-specific mCherry, use less template next time.
|
Project name
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