User:Karmella Haynes/Notebook/Polycomb project/2010/06/08

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06/08/10

  • ✓ ChIP: pull-down, wash non-specific binding & elution, DNA purification
  • ✓ ChIP Western: quality control analysis Western blot
  • ✓ Growth assay: add fresh 1 μg/mL dox to all dox+ samples



ChIP: quality control analysis Western blot
> Did chromatin-ab + bead binding for 4 hours
> Pronase treatment overnight (continue tomorrow)
> Western blot samples:

  1. mouse α-PolII ChIP Supernatant
  2. mouse α-PolII ChIP (eluted)
  3. mouse α-IgG ChIP Supernatant
  4. mouse α-IgG ChIP (eluted)
  5. Input

> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gel loading
  1. PageRuler Plus pre-stained ladder (10 μL)
  2. mouse α-PolII ChIP Supernatant (12.5 μL)
  3. mouse α-PolII ChIP (eluted) (12.5 μL)
  4. 1x loading dye
  5. mouse α-IgG ChIP Supernatant (12.5 μL)
  6. mouse α-IgG ChIP (eluted) (12.5 μL)
  7. 1x loading dye
  8. Input (12.5 μL)
  9. PageRuler
  10. 1x loading dye
6/08/10 Ponceau S stain
Ponceau S stained filter

--> Primary: mouse α-PolII (ab817), 1:1000, 5 mL

6/09/10
--> Secondary: donkey α-mouse IgG-HRP, 1:5,000 (R.T./ 1 hr.)
6/09/10 Ponceau S stain
--> PolII = 217 kD
--> Pull-down unsuccessful. Try again using more chromatin and more beads.




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