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

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07/10/10

  • ✓ Cell culture: thaw 156 lines (156-5 failed)
  • ✓ ChIP: wash & elute, Western blot



Cell culture
> Growth:

  • KAH157-1
  • 158-4
  • 159-2

--> Expand in 10 cm plates (FTRx +puro maintenance medium)

> Failed:

  • 156-5

--> Thaw: 156-2, 156-3, 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.

Gel
  1. PageRuler Plus (10 μL)
  2. myc IP sup
  3. myc IP wash 1
  4. myc IP wash 5
  5. myc IP elution
  6. IgG IP sup
  7. IgG IP wash 1
  8. IgG IP wash 5
  9. IgG IP elution
  10. 132-8 input
7/10/10 Ponceau S stain
Ponceau S stained filter

> 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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