User:Karmella Haynes/Notebook/Polycomb project/2010/06/21

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06/21/10

 * &#x2713; PCR: Amplify Pc-ATF's from 126-1 and 132-8 cDNA to verify correct transgenes
 * &#x2713; ChIP: cross-link 126-1 and 132-8 cells
 * &#x2713; Western: Sonicated chromatin (#6 132-8); new conditions for detecting modified histones
 * &#x2713; 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:
 * 1) 126-1 dox+ cDNA
 * 2) 132-8 dox+ cDNA

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.

--> Cut filters into 3 strips (2 samples, 1 ladder each) --> Block in 5% milk/PBST at R.T./ 1 hour

--> Primaries:
 * 1) rabbit α-H3 ab1791, 1:500, 3 mL
 * 2) rabbit α-H3K27me3 07-449, 1:500, 3 mL
 * 3) mouse α-H3K27me3 ab6002, 1:500, 3 mL
 * 4) rabbit α-H3K9me3 ab8898, 1:500
 * 5) rabbit α-H3K4 C42D8, 1:500, 3 mL
 * 6) 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



> Conclusions:


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