User:Karmella Haynes/Notebook/Polycomb project/2010/03/01

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03/01/10

  • ✓ Cytology: KAH130-2, 131-9, 132-8 cells (fixed last week, blocked over the w/e)
  • ✓ RNA prep: 130-2 cells, 8 day dox induction (collect in Trizol, store at -20°C)

Cytology
> See paper notebook 4/27/09
> Primary: anti-H3K27me3 (rabbit poly), 1:500
or anti-H3K9me3 (rabbit poly), 1:500
or "mock" (5% normal horse serum/ PBS, no ab)
--> R.T./ 3 hrs.
> Wash 4x with 1x PBS
> Wash 1x in 5% NHS
> Secondary: goat anti-rabbit Alexa Fluor 488, 1:1000
plus Hoescht 33325, 1:1000
--> R.T./ 1 hr.
> Wash 3x with 1x PBS
> Mount using Vinol. Let set overnight (protected from light)



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