Daniel:Notebook/HiResChrPaint/2013-12-14: Difference between revisions
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|style="font-weight:bold" width="30"| '''Step''' | |style="font-weight:bold" width="30" align="center"| '''Step''' | ||
|style="font-weight:bold" width="300"|'''Dish 3''' | |style="font-weight:bold" width="300" align="center"|'''Dish 3''' | ||
|style="font-weight:bold" width="300"|'''Dish 4''' | |style="font-weight:bold" width="300" align="center"|'''Dish 4''' | ||
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Revision as of 23:44, 13 December 2013
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 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