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:
- mouse α-PolII ChIP Supernatant
- mouse α-PolII ChIP (eluted)
- mouse α-IgG ChIP Supernatant
- mouse α-IgG ChIP (eluted)
- Input
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.
Gel loading
- PageRuler Plus pre-stained ladder (10 μL)
- mouse α-PolII ChIP Supernatant (12.5 μL)
- mouse α-PolII ChIP (eluted) (12.5 μL)
- 1x loading dye
- mouse α-IgG ChIP Supernatant (12.5 μL)
- mouse α-IgG ChIP (eluted) (12.5 μL)
- 1x loading dye
- Input (12.5 μL)
- PageRuler
- 1x loading dye
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Ponceau S stained filter
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--> 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.)
--> PolII = 217 kD
--> Pull-down unsuccessful. Try again using more chromatin and more beads.
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Project name
Main project page
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