Daniel:Notebook/RNAFISH/2015-3-4

From ZhangLabWiki
Jump to navigation Jump to search

Linnarsson Lab Protocol 2[edit]

Back to Calendar

Last week's RNA FISH protocol had too much autofluorescence (again), so I talked with Simone, who said paraformaldehyde was the biggest issue in reducing autofluorescence. He said to use freshly prepared solution, diluting it myself from 16% down to 4% and making it in PBS. Also, freezing it until ready to use. I've done all these things, so I am ready for another trial. Also, if this trial doesn't work, I can use the paraformaldehyde powder and make the whole thing myself. Finally, he said to avoid the green channel, so I will switch from using 488/594 to 546/647.

Dye Coupling[edit]

  • FOXP2-647/488
  • GAD1-546/594
  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
  5. Add 2 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
  8. Centri-Sep column purification after incubation
    1. Use TE Buffer
  9. Ethanol Precipitation; incubate overnight
  10. Continued tomorrow

Buffer Prep[edit]

  • Hybridization Buffer
    • RNAse free water 5.3 mL
    • SSC 20X 1 mL
    • Dextran sulfate 2 mL
    • Formamide 1 mL
    • E coli tRNA 500 uL
    • RVC 200 mM (warm to 37) 100 uL
    • BSA 50 mg/mL 40 uL