Daniel:Notebook/HiResChrPaint/2013-12-3
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FISH-Controls
Performing FISH on Positive Control 2 and a blank dish (I'll add DRAQ5 but no probes). The blank dish is to test DRAQ5 as well as the FISH protocol (I took pictures of the cells previously and I want to see that the cells are making it through the process (finding cells has been one of the hardest parts of FISH). The positive control will test the theory that a single color amplification will yield stronger signal and thus make a suitable positive control for the experiment (it is designed to mimic standard FISH).
Hybridization
Original Protocol from Beliveau et al: File:Interphase FISH protocol.pdf
Day 1
1. Allow stored slides to warm to room temperature 2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar 3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes 4. Remove slides and allow to cool to room temperature 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip 6. Invert slides on to cocktail-covered coverslips; 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