Daniel:Notebook/HiResChrPaint/2013-12-14: Difference between revisions

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|'''Step'''|'''Dish 3'''|'''Dish 4'''|
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|1.|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|Allow stored slides to warm to room temperature|

Revision as of 23:28, 13 December 2013

FISH Parameters

Back to Calendar

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

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