Matt:LabNotes/2015-7-22

From ZhangLabWiki
Revision as of 00:54, 23 July 2015 by >Mzcai (Created page with "=Dye coupling SNAP25 odd&even 488&594= *Combined SNAP25 odd probes (24) and even probes (24) following same protocol as [[Matt:LabNotes/2015-7-8#Combining_CUX2_odd_and_CUX2_ev...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) 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 DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
    • Dye cannot be saved for later use. Use immediately!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. 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
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight