Daniel:Notebook/HiResChrPaint/2013-8-12: Difference between revisions

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(Created page with "=FISH= Following original FISH protocol ==Slide Preparation== 1. Rinse slides in 100% ethanol; allow slides to dry completely 2. Prepare a cell ...")
 
>Djacobse
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''Day 1''
''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
  1. 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
  2. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove slides and allow to cool to room temperature
  3. Remove slides and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip
  4. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip
  6. Invert slides on to cocktail-covered coverslips; seal with rubber cement
  5. 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
  6. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  7. Denature for 2.5 minutes at 92C; keep humid
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
  8. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C

Revision as of 21:45, 12 August 2013

FISH

Following original FISH protocol

Slide Preparation

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

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