Daniel:Notebook/RNAFISH/2015-2-25

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Revision as of 18:59, 25 February 2015 by >Djacobse (Created page with "=Linnarsson Protocol (Started 2/24/2015)= Back to Calendar ==Buffer Prep== *Hybridization Buffer **RNAse f...")
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Linnarsson Protocol (Started 2/24/2015)

Back to Calendar

Buffer Prep

  • Hybridization Buffer
    • RNAse free water 5.3 mL
    • SSC 20X 1 mL
    • Dextran sulfate 2 mL
    • Formamide 1 mL
    • E coli tRNA 500 uL
    • RVC 200 mM (warm to 37) 100 uL
    • BSA 50 mg/mL 40 uL

Ethanol Precipitation

  1. Centrifuge at 4 C for 30 minutes
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge at minutes at 4 C for 10 minutes
  4. Dry the pellet in the hood; protect from light
  5. Resuspend pellet in 15 uL TE buffer

Hybridization

  1. Prepare fresh paraformaldehyde 4% and keep at RT
  2. Fix sections with 4% paraformaldehyde for 15 minutes at RT
  3. Rinse sections twice with RT PBS
  4. Permeabilize with ethanol (70% EtOH, >90 minute incuabation at 4C)
  5. Rinse sections twice with RT PBS
  6. Prepare the wash buffer and let it sit at RT
  7. Warm hybridization buffer and warm it up to 37C
  8. Prepare hybridization reaction:
    1. hybridization buffer-100 uL; probe 2-10 uL
  9. Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.