Matt:LabNotes/2015-7-14

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Revision as of 18:04, 16 July 2015 by >Mzcai (→‎Improve Dye-Coupling)
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Improve Dye-Coupling

Dan's best practice dye coupling protocol
Generic dye coupling protocol

CUX2-594 KIT-488

  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. Make 2 25ul aliquots of CUX2 48probe and KIT 48probe to prevent so many freeze-thaws in the future
  4. Denature samples for 5 minutes at 95C, then snap cool using ice box
  5. 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!
  6. Add 8 uL sample to dye tube
  7. Incubate in the dark for 1 hour
  8. Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
  9. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  10. Repeat column purification
  11. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  12. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  13. Remove supernatant and add 750 uL chilled 80% EtOH
  14. Centrifuge 5 minutes at 4 C
  15. Dry the pellet in the hood
  16. Resuspend pellet in 12 uL TE
  17. 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 = 5ul * 1ug/ul