Daniel:Notebook/HiResChrPaint/2013-12-14
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FISH Parameters
As discussed yesterday, I'm going to explore several options in the FISH protocol for potential avenues. Dr. Zhang recommended yesterday that I use 96 well plates, like Hosuk, to explore these spaces. For now, since I've got the cells, I'm going to use the Mattek dishes, but especially with option 1 there is room to explore the space. Today I'm testing the protocol using low stringency conditions for #1, and if that yields noisy signal, as expected, then the best thing to do will be to explore the parameter space using various concentrations of formamide, for which I'll use a 96 well plate.
Samples
- Dish 3: 10 pmol V6S1/V6S2; 10 pmol V6S3/V6S4; 2.5 ug human Cot1 DNA
- Dish 4: 10 pmol V6S1/V6S2; 10 pmol V6S3/V6S4; 2.5 ug human Cot1 DNA
Hybridization
Original Protocol from Beliveau et al: FISH Protocol (Beliveau)
Day 1
Dish 3|Dish 4| |
Allow stored slides to warm to room temperature|Allow stored slides to warm to room temperature|
}|
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 |