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

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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:

  1. 126-1 dox+ cDNA
  2. 132-8 dox+ cDNA
Reagent Volume Expected:
1. 126-1 = 1156
2. 132-8 = 964
6/21/10 PCR
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
  1. PageRuler Plus (10 μL)
  2. U2OS plain WCE
  3. 132-8 sonicated chromatin
  4. PageRuler Plus (10 μL)
  5. U2OS plain WCE
  6. 132-8 sonicated chromatin
  7. PageRuler Plus (10 μL)
  8. U2OS plain WCE
  9. 132-8 sonicated chromatin
  10. PageRuler Plus (10 μL)
6/21/10 Ponceau S stain
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:

  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

Image:KAH 062210 WB1.tif

> Conclusions:




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