Matt:LabNotes/2015-7-14

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Revision as of 22:28, 16 July 2015 by >Mzcai
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Improve Dye-Coupling[edit]

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
Probe ssDNA (ng/ul) 488 dye (pmol/ul) 594 dye (pmol/ul) dye:probe ratio ssDNA mass (ug) ssDNA start mass (ug) ssDNA Yield (%)
KIT-488 213.1 35.9 0.2 1.09 2.56 5 51.1
CUX2-594 233.9 -0.4 45.1 1.25 2.81 5 56.1

Conclusion[edit]

  • Alexa594 is significantly larger, more hydrophobic, and less charged than Alexa 488
    • Therefore possible size exclusion column is not as effective at removing it
    • And more likely to precipitate?
    • Try using PCR purification column instead of Centri-Sep column
  • Use Alexa647 instead (with Alexa488)
    • Dr. Bern's microscope does not have far red filter
    • Must use Dr. Wang's Nikon II microscope (doesn't have DAPI)
    • See if Dr. Huang's lab has FITC, Cy5, and DAPI filters with >60X Obj and 1.4NA and <-20C CCD