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
Step | Dish 3 | Dish 4 |
1A. | Allow stored slides to warm to room temperature | Allow stored slides to warm to room temperature |
1A. | Incubate 30 minutes in 70% Ethanol | |
1B. | Incubate 1 minutes in 0.1N 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 |