Matt:LabNotes/2015-6-15
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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
- Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
- Add 5 uL DNA (~1ug/ul) 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 DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
- Dye cannot be saved for later use. Use immediately!
- Add 8 uL sample to dye tube
- Incubate in the dark for 1 hour
- Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- No vacufuge
- Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight