User:Karmella Haynes/Notebook/Polycomb project/2010/08/20

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08/20/10

 * &#x2713; ChIP/ Co-IP: KAH126-1 and 132-8

ChIP: myc-bead pull-down > See 6/29/10 > Prep sonicated ~6 mL samples according to Qingqing's protocol --> Add: --> Save 4x 100 μL samples as "Input"
 * 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: --> Rotate at 4°C overnight --> Wash and elute tomorrow
 * 1) KAH126-1: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 2) KAH126-1: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
 * 3) KAH132-8: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 4) KAH132-8: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

8/21/10 > 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 50 μL supernatant. Discard remaining sup. --> Wash beads in 500 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat twice more (3 washes total) --> Add 25 μL 4x loading dye (+100 mM DDT) and 25 μL RIPA (lysis/ protein dilution buffer) to each pellet. Heat at 100°C/ 5 min., vortex, repeat. --> Clear supernatant by spinning at 4000 rpm/ 2 min. Transfer 10 μL 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)


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