06/21/10
- ✓ PCR: Amplify Pc-ATF's from 126-1 and 132-8 cDNA to verify correct transgenes
- ✓ ChIP: cross-link 126-1 and 132-8 cells
- ✓ Western: Sonicated chromatin (#6 132-8); new conditions for detecting modified histones
- ✓ Cell culture: thaw 127-4, 128-8.3, 129-4, 130-2, 130-7, 131-1, 133-1
PCR
> Primers: CMVTetO-r2-rc/ Myc r1
> Templates:
- 126-1 dox+ cDNA
- 132-8 dox+ cDNA
| Reagent |
Volume
|
Expected:
1. 126-1 = 1156
2. 132-8 = 964
|
5 μL/lane; 1% agarose
|
| cDNA |
0.5
|
| 10 μM primer mix |
1.0
|
| 2x GoTaq |
12.5
|
| dH2O |
11.0
|
| |
25.0 μL
|
PCR: DNA engine block A
- 95°C/ 3 min.
- [95°C/ 30 sec.; 57°C/ 30 sec.; 72°C/ 30 sec.] x30
- 72°C/ 3 miin.
- 4°C/ ∞
--> Conclusions: sizes look correct
Western blot
> Consulted Steve from Cell Signaling Technology
- Blot soluble chromatin or sonicated whole cell lysate, not clarified whole cell lysate (chromatin ends up in the discarded pellet)
- Be sure nitrocellulose pore size is small (0.2 μm is good)
- Probing conditions: Block with 5% milk, primary stain with 5% BSA, secondary stain with 5% milk
> Gels (duplicates)
--> Samples loaded (in replicate):
- U2OS plain whole cell extract (43.1 μg, 18.75 μL)
- 132-8 sonicated chromatin (18.75 μL)
> Use 4x loading dye w/ BME (from Qingqing & Mike)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.
Gel
- PageRuler Plus (10 μL)
- U2OS plain WCE
- 132-8 sonicated chromatin
- PageRuler Plus (10 μL)
- U2OS plain WCE
- 132-8 sonicated chromatin
- PageRuler Plus (10 μL)
- U2OS plain WCE
- 132-8 sonicated chromatin
- PageRuler Plus (10 μL)
|
Ponceau S stained filters
|
--> Cut filters into 3 strips (2 samples, 1 ladder each)
--> Block in 5% milk/PBST at R.T./ 1 hour
--> Primaries:
- rabbit α-H3 ab1791, 1:500, 3 mL
- rabbit α-H3K27me3 07-449, 1:500, 3 mL
- mouse α-H3K27me3 ab6002, 1:500, 3 mL
- rabbit α-H3K9me3 ab8898, 1:500
- rabbit α-H3K4 C42D8, 1:500, 3 mL
- mouse α-PolII 8WG16, 1:1000, 3 mL
6/22/10
--> Secondaries (R.T./ 1 hr.):
1,2,4,5. donkey α-rabbit IgG-HRP, 1:5000, 3 mL
3,6. donkey α-mouse IgG-HRP, 1:5000, 3 mL
6/22/10 Western blot
> Conclusions:
|
Project name
Main project page
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