Daniel:Notebook/RNAFISH/2015-3-5

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Revision as of 17:27, 5 March 2015 by >Djacobse (→‎Ethanol Precipitation)
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Linnarsson Lab Protocol 2 (Started 3/4/2015)

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Dye Coupling (Continued; Ethanol Precipitation)

  1. Remove from -80C
  2. Centrifuge at 4 C at 12000 rpm for 30 minutes
  3. Remove supernatant and add 700 uL chilled 75% EtOH
  4. Centrifuge at minutes at 4 C for 10 minutes at 12000 rpm
  5. Remove supernatant and dry the pellet in the hood
  6. Resuspend pellet in 7.5 uL TE buffer
  7. Store DNA at 4C for immediate use

Nanodrop Results

Hybridization

Sample Matrix

  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. Permeabilization-may use:
    1. 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.