Matt:LabNotes/2015-7-22
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Dye coupling SNAP25 odd&even 488&594
- Combined SNAP25 odd probes (24) and even probes (24) following same protocol as CUX2 odd&even
- SNAP25_odd: 1148.3 ng/ul
- SNAP25_even: 1154.0 ng/ul
Dye coupling
Dan's best practice dye coupling protocol
Generic dye coupling protocol
- SNAP25_odd-488
- SNAP25_even-594
- 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 H2O to sample
- Previously did 80ul TE but maybe H2O will work better because no amine groups
- 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