Matt:LabNotes/2015-6-15

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Revision as of 23:54, 15 June 2015 by >Mzcai (Created page with "==Motor Neurons RNA FISH Dye Coupling== *Matt:LabNotes/2015-6-8#DARTFISH ===Dye Coupling=== [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coup...")
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Motor Neurons RNA FISH Dye Coupling

Dye Coupling

Dan's best practice dye coupling protocol
Generic dye coupling protocol

  • Sebastian said in email the two highest expressed genes from our 48 probe set of genes are CUX2 and KIT
  • I am going to try both 488/594 and 546/647 pairs of fluorophores in case one set is not compatible with our Olympus filter cubes

CUX2-488 KIT-594 CUX2-546 KIT-647

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) 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 DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
    • Dye cannot be saved for later use. Use immediately!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight