Daniel:Notebook/HiResChrPaint/2014-8-5
Jump to navigation
Jump to search
No Wash Hybridization
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
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C