Daniel:Notebook/RNAFISH/2015-3-4
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Linnarsson Lab Protocol 2[edit]
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
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- Add 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
- Centri-Sep column purification after incubation
- Use TE Buffer
- Ethanol Precipitation; incubate overnight
- 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