Matt:LabNotes/2015-7-14: Difference between revisions
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==Conclusion== | |||
*[https://www.lifetechnologies.com/order/catalog/product/A20004 Alexa594] is significantly larger, more hydrophobic, and less charged than [https://www.lifetechnologies.com/order/catalog/product/A20000 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 |
Latest revision as of 22:28, 16 July 2015
Improve Dye-Coupling[edit]
Dan's best practice dye coupling protocol
Generic dye coupling protocol
CUX2-594 KIT-488
- 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
- Make 2 25ul aliquots of CUX2 48probe and KIT 48probe to prevent so many freeze-thaws in the future
- 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 1xTE to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- No vacufuge
- Repeat column purification
- 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 80% 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 = 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