Daniel:Notebook/HiResChrPaint/2013-10-18
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FISH[edit]
Trying FISH again to also view under the confocal. I'll use BSPP-2 Control, and in the sample I'll use V6S2 and V6S3.
- Control: 20 pmol BSPP-2, 2.5 ug cot-1 DNA
- Experiment: 4 uL (10 pmol) V6S3, 2 uL (10 pmol) V6S2, 2.5 ug cot-1 DNA
Hybridization[edit]
Protocol[edit]
- Modified from standard FISH protocol
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; prewarm formamide 3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes 4. Remove media and allow slides to cool to room temperature 5. Add 25 uL hybridization cocktail to center of dish 6. Apply a 22x22 #1.5 cover slip to center of dish; 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 42C