Daniel:Notebook/RNAFACS/2016-1-12

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Poly T Primer Test (Started 01/11/16)[edit]

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Buffers[edit]

  • Wash Buffer A
    • 200 uL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60)
    • Add 700 uL Nuclease-free water
    • Add 100 uL Deionized Formamide
    • Mix well by vortexing gently
  • Wash Buffer A w/DAPI
    • 200 uL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60)
    • Add 700 uL Nuclease-free water
    • Add 100 uL Deionized Formamide
    • Add 1 uL of 20 ug/mL DAPI stock (dilute original 1:1000 and store in 4C)
    • Mix well by vortexing gently

Protocol[edit]

  1. Aspirate out hybridization buffer and add 1 mL of Wash Buffer A
  2. Incubate in the dark at 37 °C for 30 minutes
  3. Aspirate the Wash Buffer A, and then add 1 mL of DAPI nuclear stain (Wash Buffer A consisting of 5 ng/mL DAPI) to counterstain the nuclei
  4. Incubate in the dark at 37 °C for 30 minutes
  5. Aspirate the DAPI staining buffer, and then add 1 mL of Wash Buffer B. Incubate at room temperature for 2-5 minutes
  6. Add a small drop (approximately 15 μL) of Vectashield Mounting Medium onto center of dish
  7. Seal the coverglass perimeter with clear nail polish, and allow to dry
  8. If necessary, gently wipe away any dried salt off the coverglass with water