Daniel:Notebook/HiResChrPaint/2013-9-27: Difference between revisions

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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.
'''Samples''':
*10 pmol [[Daniel:Notebook/HiResChrPaint/2013-7-17#Labeling Results|V6S3]], 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-7-25#Labeling Results|V4S2]], 2.5 ug human Cot-1 DNA;
*20 pmol [[Daniel:Notebook/HiResChrPaint/2013-9-5|BSPP-1-546]], 2.5 ug human Cot-1 DNA


==FISH Protocol==
==FISH Protocol==


*Following [[Daniel:Protocols/FISH#Hybridization|standard FISH procedure]]
*Following modified [[Daniel:Protocols/FISH#Hybridization|FISH procedure]]


''Day 1''
''Day 1''
  1. Allow stored slides to warm to room temperature
  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
  2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C; use pre-warmed solution
  3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove slides and allow to cool to room temperature
  4. Remove media from slides and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip (¿add to center?)
  5. Add 25 uL hybridization cocktail to center of slide
  6. Invert slides on to cocktail-covered coverslips; seal with rubber cement (¿Seal dish with parafilm?)
  6. Denature for 2.5 minutes at 92C; keep humid; used rock bath
7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  7. Transfer slides to a humidified chamber and hybridize overnight at 42C
8. Denature for 2.5 minutes at 92C; keep humid
Continued [[Daniel:Notebook/HiResChrPaint/2013-9-28|tomorrow]]
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C

Latest revision as of 16:56, 30 September 2013

FISH[edit]

Back to Calendar

Using the slides from Tuesday, I'm going to do another FISH experiment.

Samples:

  • 10 pmol V6S3, 10 pmol V4S2, 2.5 ug human Cot-1 DNA;
  • 20 pmol BSPP-1-546, 2.5 ug human Cot-1 DNA

FISH Protocol[edit]

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; use pre-warmed solution
3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
4. Remove media from slides and allow to cool to room temperature
5. Add 25 uL hybridization cocktail to center of slide
6. Denature for 2.5 minutes at 92C; keep humid; used rock bath
7. Transfer slides to a humidified chamber and hybridize overnight at 42C
Continued tomorrow