User:Karmella Haynes/Notebook/Polycomb project/2010/10/01

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10/01/10

 * &#x2713; ChIP/ Co-IP: IP for KAH126-1, 132-8, and 130-4 chromatin (for Western)
 * &#x2713; RT-PCR: Pc-ATF biological rep's, redo p16 (2x)
 * &#x2713; Cell culture: expand HEK Gal4-EED to two 75 cm2 flasks (2 μg/mL puro)

RT-PCR > Pc-ATF biological replicates (30 total) --> Templates (6/15, 6/18, 6/23/10) --> Primers
 * 1) KAH126-1 -dox
 * 2) KAH126-1 +dox
 * 3) KAH126-3 -dox
 * 4) KAH126-3 +dox
 * 5) KAH132-8 -dox
 * 6) KAH132-8 +dox
 * 7) KAH154-2 -dox
 * 8) KAH154-2 +dox
 * 9) FTRx -dox
 * 10) FTRx +dox
 * 1) p16INK 7C (94 bp), (1.0 μL 1:1 cDNA) (30 rxns)

--> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
 * 72°C/ 3 min.
 * 4°C/ ∞



> Conclusions:

ChIP: myc-bead pull-down > See 9/15/10 > Sonicated ~6 mL samples prepped according to Qingqing's protocol --> Added:
 * 720 μL 10% Triton X-100
 * 72 μL 10% Na-deoxycholate (DOC)
 * 500 μL TE (pH 8.0)
 * 6 μL 1000x PLAAC
 * 60 μL 100x PMSF

> Binding (3 each): --> Rotate at 4°C >overnight
 * 1) KAH130-4: 400 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 2) KAH130-4: 400 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
 * 3) KAH132-8: 400 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 4) KAH132-8: 400 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
 * 5) KAH126-1: 400 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 6) KAH126-1: 400 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

10/03/10 > Wash & elute IP's (Keep everything on ice ) --> Prepare washing buffer (complete sonication buffer): --> Spin down beads at 3000 rpm/ 3 min./ 4°C --> Save 100 μL Supernatant. Discard remaining sup. --> Wash beads in 400 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat three more times (4 washes total) --> Add 60 μL 1x loading dye (50 uL 4x l.d. + 150 RIPA, ~50 mM DTT) to each pellet. Heat at 100°C/ 5 min., vortex. --> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube (save at -20°C for Western later).
 * 6 mL Buffer III
 * 60 μL 100x PMSF
 * 6 μL 1000x PLAAC
 * 720 μL 10% Triton X-100
 * 72 μL 10% Na-deoxycholate (DOC)
 * 500 μL TE (pH 8.0)

> α-myc bead control for Western: --> Add 400 μL wash buffer to 10 μL α-myc beads. Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. --> Add 30 μL 1x loading dye (250 uL 4x l.d. + 750 dH2O, + ~50 mM DTT) to each pellet. Heat at 100°C/ 5 min., vortex. --> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube (save at -20°C for Western later).


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