Daniel:Notebook/HiResChrPaint/2014-8-5

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Revision as of 20:49, 5 August 2014 by >Djacobse (Created page with "=No Wash Hybridization= Back to Calendar I'll be continuing my FISH experiments with a modified protocol. I'm going to skip the wash steps...")
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No Wash Hybridization

Back to Calendar

I'll be continuing my FISH experiments with a modified protocol. I'm going to skip the wash steps, fearing that most probe is being washed away during these phases. Alternatively, I'm going to explore a change in the protocol. I found in a more in depth Beliveau publication that the oligos are not resuspended, for instance after vacuum centrifuge like I have been doing, but are rather added in a concentrated fashion to the hybridization mix, usually ≤4 uL.

Hybridization

Sample Matrix


  1. Allow stored samples to warm to room temperature
  2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; seal with rubber cement
  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C