Daniel:Notebook/HiResChrPaint/2013-12-3: Difference between revisions

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=FISH-Controls=
=FISH-Controls=
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]


Performing FISH on [[Daniel:Notebook/HiResChrPaint/2013-11-13#Positive Control 2: Single Color Amplification|Positive Control 2]] and a blank dish (I'll add DRAQ5 but no probes).  The blank dish is to test DRAQ5 as well as the FISH protocol (I took pictures of the cells [[Daniel:Notebook/HiResChrPaint/2013-11-29|previously]] and I want to see that the cells are making it through the process (finding cells has been one of the hardest parts of FISH).  The positive control will test the theory that a single color amplification will yield stronger signal and thus make a suitable positive control for the experiment (it is designed to mimic standard FISH).
Performing FISH on [[Daniel:Notebook/HiResChrPaint/2013-11-13#Positive Control 2: Single Color Amplification|Positive Control 2]] and a blank dish (I'll add DRAQ5 but no probes).  The blank dish is to test DRAQ5 as well as the FISH protocol (I took pictures of the cells [[Daniel:Notebook/HiResChrPaint/2013-11-29|previously]] and I want to see that the cells are making it through the process (finding cells has been one of the hardest parts of FISH).  The positive control will test the theory that a single color amplification will yield stronger signal and thus make a suitable positive control for the experiment (it is designed to mimic standard FISH).
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''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 in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  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 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 center of dish
6. Invert slides on to cocktail-covered coverslips; seal with rubber cement
6. Cover with 22x22 #1.5 coverslip, seal with rubber cement
  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 water bath and hybridize overnight at 42C

Latest revision as of 23:14, 4 December 2013

FISH-Controls[edit]

Back to Calendar

Performing FISH on Positive Control 2 and a blank dish (I'll add DRAQ5 but no probes). The blank dish is to test DRAQ5 as well as the FISH protocol (I took pictures of the cells previously and I want to see that the cells are making it through the process (finding cells has been one of the hardest parts of FISH). The positive control will test the theory that a single color amplification will yield stronger signal and thus make a suitable positive control for the experiment (it is designed to mimic standard FISH).

  • BLANK sample: 0 pmol probe in hybridization cocktail
  • Positive Control: 10 pmol V6S1/V6S2; 10 pmol V6S3/V6S4; 2.5 ug human Cot1 DNA

Hybridization[edit]

Original Protocol from Beliveau et al: File:Interphase FISH protocol.pdf

Day 1

1. Allow stored slides to warm to room temperature
2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
3. Incubate in 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 center of dish
6. Cover with 22x22 #1.5 coverslip, seal with rubber cement
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 water bath and hybridize overnight at 42C