Daniel:Notebook/HiResChrPaint/2013-12-14

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Revision as of 23:42, 13 December 2013 by >Djacobse (→‎Hybridization)
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FISH Parameters

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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

Hybridization

Original Protocol from Beliveau et al: FISH Protocol (Beliveau)

Day 1

Step Dish 3 Dish 4
1A. Allow stored slides to warm to room temperature Allow stored slides to warm to room temperature
1A.   Incubate 10 minutes in 70% Ethanol
1B.   Incubate 10 minutes in 0.1M HCl
2. Incubate slides in 2X SSCT +25% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
3. Incubate in 2X SSCT + 25% formamide at 60C for 20 minutes Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
4. Remove media and allow to cool to room temperature Remove media and allow to cool to room temperature
5. Add 25 uL hybridization cocktail to center Add 25 uL hybridization cocktail to center
6. Add a 22x22#1.5 coverslip to center of dish; seal with rubber cement Add a 22x22#1.5 coverslip to center of dish; seal with rubber cement
7. Allow the rubber cement to air-dry for 5 minutes at room temperature Allow the rubber cement to air-dry for 5 minutes at room temperature
8. Denature for 2.5 minutes at 92C; keep humid Denature for 2.5 minutes at 92C; keep humid
9. Transfer slides to a humidified chamber and hybridize overnight at 37C Transfer slides to a humidified chamber and hybridize overnight at 37C


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