09/13/10
- ✓ RNA prep/ cDNA: p16 induction time course
- ✓ ChIP/ Co-IP: KAH130-4 sample
- ✓ Midipreps: Use QIAfilter Plasmid Midi kit (100 mL culture, used Maxi volumes to prep and ppt lysate, midi volumes to wash/ elute/ ppt DNA)
- ✓ Culture: HEK cells failed to grow; get back-up vial from Mirra
RNA prep
> Use Qiagen QIAshredder and RNeasy kit (no BME added to lysis buffer)
> Elute with 50 μL RNase-free H2O
| Sample |
A260 |
260/280 |
ng/ul |
uL = 2ug RNA |
dH2O
|
| 1. KAH126-1, 1-day |
17.289 |
2.08 |
691.58 |
2.9 |
5.1
|
| 2. KAH126-1, 2-day |
16.842 |
2.12 |
673.67 |
3.0 |
5.0
|
| 3. KAH126-1, 3-day |
17.923 |
2.06 |
716.92 |
2.8 |
5.2
|
| 4. KAH126-1, 4-day |
15.9 |
2.05 |
635.99 |
3.1 |
4.9
|
| 5. KAH154-2, 1-day |
23.301 |
2.07 |
932.03 |
2.1 |
5.9
|
| 6. KAH154-2, 2-day |
16.135 |
2.09 |
645.41 |
3.1 |
4.9
|
| 7. KAH154-2, 3-day |
18.431 |
2.08 |
737.24 |
2.7 |
5.3
|
| 8. KAH154-2, 4-day |
13.603 |
2.08 |
544.11 |
3.7 |
4.3
|
| 9. KAH132-8, 1-day |
18.812 |
2.07 |
752.49 |
2.7 |
5.3
|
| 10. KAH132-8, 2-day |
15.47 |
2.06 |
618.8 |
3.2 |
4.8
|
| 11. KAH132-8, 3-day |
13.727 |
2.08 |
549.07 |
3.6 |
4.4
|
| 12. KAH132-8, 4-day |
10.627 |
2.01 |
425.07 |
4.7 |
3.3
|
| 13. FTRx, 1-day |
20.22 |
2.07 |
808.8 |
2.5 |
5.5
|
| 14. FTRx, 2-day |
22.792 |
2.08 |
911.69 |
2.2 |
5.8
|
| 15. FTRx, 3-day |
22.614 |
2.09 |
904.55 |
2.2 |
5.8
|
| 16. FTRx, 4-day |
14.333 |
2.07 |
573.32 |
3.5 |
4.5
|
--> Store remainder of RNA at -80°C
7/26/10
> 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
--> 16 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 0.8 μL RNase H, 37°C/ 20 min.
--> Store at -20°C
ChIP: myc-bead pull-down
> See 6/29/10
> Prep sonicated ~6 mL samples according to Qingqing's protocol
--> Add:
- 720 μL 10% Triton X-100
- 72 μL 10% Na-deoxycholate (DOC)
- 500 μL TE (pH 8.0)
- 6 μL 1000x PLAAC
- 60 μL 100x PMSF
--> Save 4x 100 μL samples as "Input"
> Binding:
- KAH130-4: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
- KAH130-4: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
--> Rotate at 4°C overnight
--> Wash and elute tomorrow
9/14/10
> Wash & elute IP's (Keep everything on ice)
--> Prepare washing buffer (complete sonication buffer):
- 6 mL Buffer III
- 60 μL 100x PMSF
- 6 μL 1000x PLAAC
- 720 μL 10% Triton X-100
- 72 μL 10% Na-deoxycholate (DOC)
- 500 μL TE (pH 8.0)
--> Spin down beads at 3000 rpm/ 3 min./ 4°C
--> Save 50 μL supernatant. Discard remaining sup.
--> Wash beads in 500 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat three more times (4 washes total)
--> Add 60 μL 1x loading dye (50 uL 4x l.d. + 150 RIPA, ~50 mM DTT) to each pellet. Heat at 100°C/ 5 min., vortex.
--> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube (save at -20°C for Western later).
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