Matt:LabNotes/2015-7-14: Difference between revisions
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>Mzcai (Created page with "=Improve Dye-Coupling= Dan's best practice dye coupling protocol<br> [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye ...") |
>Mzcai mNo edit summary |
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[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] | [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] | ||
CUX2-594 | CUX2-594 | ||
KIT-488 | KIT-488 | ||
#Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature | #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 | #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 | #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 | #Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing | ||
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#*Use 1X TE Buffer | #*Use 1X TE Buffer | ||
#*No vacufuge | #*No vacufuge | ||
#Repeat column purification | |||
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight<!-- | #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) | #Centrifuge at 4 C for 30 minutes (14,000 rpm) |
Revision as of 18:27, 15 July 2015
Improve Dye-Coupling
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