Daniel:Notebook/HiResChrPaint/2013-8-12

From ZhangLabWiki
Jump to navigation Jump to search

FISH[edit]

Back to Calendar

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