Daniel:Notebook/HiResChrPaint/2014-7-19

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Dystrophin Probes (Started 7/18/2014)[edit]

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Dye Labeling[edit]

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 2 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. Recommend column purification after incubation

Labeling Results[edit]

Sample ng/uL ssDNA pmol/uL dye pmol/uL ssDNA dye/probe bp:dye ratio
Dystrophin-488 23.3 3.9 3.5 1.1 18
Dystrophin-546 25.2 5.5 3.8 1.4 14

Good labeling results. Proceeding to FISH.

FISH Hybridization[edit]

Sample Matrix

Sample Cot1-488 (ug) Dystrophin-488 (pmol) Dystrophin-546 (pmol) Cot1 (ug)
A 0 8.6 (30) 0 2.5 (2.5)
B 0 0 7.9 (30) 2.5 (2.5)
C 5 (0.4) 0 0 0
  1. Allow stored samples 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 samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 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 a humidified chamber and hybridize overnight at 42C