User:Karmella Haynes/Notebook/Polycomb project/2010/07/10

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07/10/10

 * &#x2713; Cell culture: thaw 156 lines (156-5 failed)
 * &#x2713; ChIP: wash & elute, Western blot

Cell culture > Growth: --> Expand in 10 cm plates (FTRx +puro maintenance medium)
 * KAH157-1
 * 158-4
 * 159-2

> Failed: --> Thaw: 156-2, 156-3, 156-5
 * 156-5

ChIP: wash, elute, Western blot > Wash & elute IP (Keep everything on ice ) --> Prepare washing buffer (6 mL complete sonication buffer plus PLAAC, PMSF, detergent, salts, etc.). --> Spin down beads (3000 rpm/ 4°C/ 3 min.). --> Save 50 μL supernatant. Discard remaining sup. Wash beads in 500 μL wash buffer. Spin. Save 50 μL wash 1 sup. --> Discard sup. Wash beads in 500 μL wash buffer. Repeat 3 more times. Save 50 μL wash 5 sup. --> Add 10 μL 4x loading dye to each bead pellet. Heat at 100°C/ 5 min., vortex, heat again. --> Clear supernatant by spinning at 4000 rpm/ 2 min. Transfer 10 μL sup to new tube (for Western).

> Western blot loading > Sup, wash, & input samples: 18.75 protein + 6.25 4x buffer > Use 4x loading dye (Invitrogen) w/ BME (400 μL loading dye + 40 μL BME) > Use 10-well gel (loading volume = 25 μL) > Electroblot: 1 hr. 15 min.

> Block: 5% milk/PBST, R.T./1 hr. > Primary staining: 5% BSA/PBST, 4°C/o.n. --> rabbit α-DsRed 632496, 1:1000, 5 mL

7/11/10 > Secondary staining: 5% milk/PBST, R.T./ 1 hr. --> donkey α-rabbit, 1:5000, 5 mL


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