Daniel:Notebook/RNAFACS/2016-1-6

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Poly T/Stellaris[edit]

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Today I'll be starting my test of the poly T probes. These should work in any cell with RNA, and I should see pretty strong signal. Hopefully this gives me a nice positive result. The first thing I am going to do is test the poly T probe (alone) using the Stellaris protocol. The first step in this is getting cells, which Yan has been growing. 293T cells, for the record.

Cell Adhesion and Fixation[edit]

  1. Adhere cells to center of Mattek dish
    1. When passaging, use approximately 20 uL cells and 80 uL growth media
    2. Incubate at 37C for 3-6 hours to allow cells to adhere properly
  2. Aspirate growth media and wash with 1 mL 1X PBS
  3. Add 1 mL fixation buffer (3.7% formaldehyde in 1X PBS) and incubate 10 min at RT
  4. Wash twice with 1 mL 1X PBS
  5. Add 1 mL 70% EtOH and incubate for at least 1 hour at 4C. May incubate up to a week.