Daniel:Notebook/HiResChrPaint/2013-8-12
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FISH[edit]
FISH Slides/Hybridization Cocktails[edit]
1. Control: No DNA, vacuum centrifuged 4 uL nfH20
2. V4S2S3: 2 uL V4S2 and 2 uL V4S3
3. V6S2S3: 2 uL V6S2 and 2 uL V6S3
4. V6S2S3Cot-1: 2 uL V6S2 and 2 uL V6S3 + 2.5 ug Cot-1 DNA
1. Combine probes into 1.5 mL eppendorf tube and dry in a vacuum centrifuge 2. Resuspend DNA in 12.5 uL 100% formamide; mix at 37C for at least 10 minutes 3. Add 12.5 uL 4X SSCT, 20% w/v dextran sulfate, 10 ug RNAase A
Following original FISH protocol
Slide Preparation[edit]
1. Rinse slides in 100% ethanol; allow slides to dry completely 2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media 3. Add 100 uL of cell suspension per slide roughly in the center 4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line 5. Rinse slides briefly in 1X PBS 6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS 7. Rinse briefly in 1X PBS in a coplin jar 8. Incubate for 5 minutes in 2X SSCT 9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide 10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide
Hybridization[edit]
Day 1
1. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar 2. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes 3. Remove slides and allow to cool to room temperature 4. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip 5. Invert slides on to cocktail-covered coverslips; seal with rubber cement 6. Allow the rubber cement to air-dry for 5 minutes at room temperature 7. Denature for 2.5 minutes at 92C; keep humid 8. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C