Matt:LabNotes/2015-7-22
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Dye coupling SNAP25 odd&even 488&594[edit]
Combine SNAP25_odd and SNAP25_even[edit]
- 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[edit]
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 7.5 uL DNA (~1ug/ul) and 4.5 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 (Mixed 12ul but after heating only ~8ul left)
- 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
- Centrifuge at 4 C for 30 minutes (14,000 rpm)
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge 5 minutes at 4 C
- Dry the pellet in the hood
- Resuspend pellet in 12 uL TE
- Check dye:probe ratio with Nanodrop
- Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
- Starting DNA mass = 7.5ul * 1ug/ul
Probe | ssDNA (ng/ul) | 488 dye (pmol/ul) | 594 dye (pmol/ul) | dye:probe ratio | ssDNA mass (ug) | ssDNA start mass (ug) | ssDNA Yield (%) |
SNAP25_odd-488 | 413.9 | 66.1 | -0.1 | 1.04 | 5.0 | 7.5 | 66.667 |
SNAP25_even-594 | 455.3 | -1.2 | 89 | 1.27 | 5.5 | 7.5 | 73.333 |
SNAP25_even-594 | 477.3 | -1.4 | 88.3 | 1.20 | 5.7 | 7.5 | 76 |