User:Karmella Haynes/Notebook/Polycomb project/2010/06/11

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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06/11/10

 * &#x2713; ChIP: bead binding, bead wash, elution, quality control Western
 * &#x2713; Cell culture: split 126-1, 126-3, 132-8, 154-2, FTRx 1:10
 * &#x2713; ChIP cell cultures: 5x 25 ml plates for 126-1, 154-2, 132-8, FTRx (+ 1μg/mL dox)
 * &#x2713; RT-PCR: start 6-well plates w/ 126-1, 126-4, 132-8, 154-2, FTRx -/+ 1μg/mL dox; seeded ~250k cells
 * &#x2713; Plate for Pc-ATF Western: 126-1, 132-8, 154-2 -/+ 1μg/mL dox; seeded ~250k cells

Remember to refresh dox every two days

ChIP quality control Western > Pronase treatment overnight (continue tomorrow) > Western blot samples: > Use 10-well gel (loading volume = 25 μL) > Electroblot: 1 hr. 30 min.
 * 1) 126-1 mouse α-PolII ChIP Supernatant
 * 2) 126-1 mouse α-PolII ChIP Eluate
 * 3) 126-1 rabbit α-myc ChIP Supernatant
 * 4) 126-1 rabbit α-myc ChIP Eluate
 * 5) 126-1 rabbit α-H3K27me3 ChIP Supernatant
 * 6) 126-1 rabbit α-H3K27me3 ChIP Eluate
 * 7) 126-1 mouse/rabbit α-IgG ChIP Supernatant
 * 8) 126-1 mouse/rabbit α-IgG ChIP Eluate
 * 9) 126-1 Input
 * 10) U2OS mouse α-PolII ChIP Supernatant
 * 11) U2OS mouse α-PolII ChIP Eluate
 * 12) U2OS rabbit α-myc ChIP Supernatant
 * 13) U2OS rabbit α-myc ChIP Eluate
 * 14) U2OS rabbit α-H3K27me3 ChIP Supernatant
 * 15) U2OS rabbit α-H3K27me3 ChIP Eluate
 * 16) U2OS mouse/rabbit α-IgG ChIP Supernatant
 * 17) U2OS mouse/rabbit α-IgG ChIP Eluate
 * 18) U2OS Input

Note: Not very much protein. Grow new 25 mL plates for fresh chromatin preps.

--> Cut filters into strips, group by antibody --> Block overnight (or longer)

6/13/10 --> Primaries: 1,5. mouse α-PolII 8WG16 ab817, 1:500, 2 mL 2,6. rabbit α-myc ab9106, 1:1000, 2 mL 3,7. rabbit α-H3K27me3 07-449, 1:500, 2 mL 4,8. mouse/rabbit α-IgG sc2025/sc2027 (hold, wait for results from other blots)

6/14/10 --> Secondaries (R.T./ 1 hr.): 1,5. donkey α-mouse IgG-HRP, 1:5,000 2,6. donkey α-rabbit IgG-HRP, 1:5,000 3,7. donkey α-rabbit IgG-HRP, 1:5,000



--> Pull-down unsuccessful. Work with Qingqing to optimize procedure.


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