Daniel:Notebook/HiResChrPaint/2014-2-23: Difference between revisions

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#Allow the rubber cement to air-dry for 5 minutes at room temperature
#Allow the rubber cement to air-dry for 5 minutes at room temperature
#Denature for 2.5 minutes at 92C; keep humid
#Denature for 2.5 minutes at 92C; keep humid
#Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
#Transfer slides to a humidified chamber and hybridize overnight at 42C

Revision as of 19:54, 23 February 2014

Multi-Color FISH (Started 02/08/2014)

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Hybridization

Sample Matrix

Sample Sample A uL sample A (pmol) Sample B uL sample B (pmol)
V6S3 Only V6S3-546 5 (10) Unlabeled Cot1 1 (1 ug)
V6S3+Cot1 V6S3-546 5 (10) Cot1-488 6 (10)
Multi-Color Cot1 Cot1-High-546 20 (4) Cot1-Low-488 5 (5)
BSPP Negative Control BSPP1 5 (10) Unlabeled Cot1 1 (1 ug)

Day 1

  1. Mix sample probes and vacuum centrifuge
  2. Allow stored samples to warm to room temperature
  3. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  4. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  5. Remove samples and allow to cool to room temperature
  6. Add 25 uL hybridization cocktail to center
  7. Add 22x22 #1.5 coverslip; seal with rubber cement
  8. Allow the rubber cement to air-dry for 5 minutes at room temperature
  9. Denature for 2.5 minutes at 92C; keep humid
  10. Transfer slides to a humidified chamber and hybridize overnight at 42C