Daniel:Notebook/HiResChrPaint/2013-9-27: Difference between revisions
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>Djacobse (Created page with "=FISH= Back to Calendar Using the slides from Tuesday, I'm going to do ano...") |
>Djacobse (→FISH) |
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Using the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|Tuesday]], I'm going to do another FISH experiment. | Using the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|Tuesday]], I'm going to do another FISH experiment. | ||
==FISH Protocol== | |||
*Following [[Editing Daniel:Protocols/FISH#Hybridization|standard FISH procedure]] | |||
''Day 1'' | ''Day 1'' | ||
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3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes | 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 | 4. Remove slides and allow to cool to room temperature | ||
5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip | 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip (¿add to center?) | ||
6. Invert slides on to cocktail-covered coverslips; seal with rubber cement | 6. Invert slides on to cocktail-covered coverslips; seal with rubber cement (¿Seal dish with parafilm? | ||
7. Allow the rubber cement to air-dry for 5 minutes at room temperature | 7. Allow the rubber cement to air-dry for 5 minutes at room temperature | ||
8. Denature for 2.5 minutes at 92C; keep humid | 8. Denature for 2.5 minutes at 92C; keep humid | ||
9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C | 9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C |
Revision as of 18:36, 27 September 2013
FISH
Using the slides from Tuesday, I'm going to do another FISH experiment.
FISH Protocol
- Following standard FISH procedure
Day 1
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 (¿add to center?) 6. Invert slides on to cocktail-covered coverslips; seal with rubber cement (¿Seal dish with parafilm? 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