Matt:LabNotes/2015-7-14: Difference between revisions
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#*No vacufuge | #*No vacufuge | ||
#Repeat column purification | #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) | ||
#Remove supernatant and add 750 uL chilled 80% EtOH | #Remove supernatant and add 750 uL chilled 80% EtOH | ||
Line 27: | Line 27: | ||
#Check dye:probe ratio with Nanodrop | #Check dye:probe ratio with Nanodrop | ||
#*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) | #*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) | ||
#*Starting DNA mass = 5ul * 1ug/ul | #*Starting DNA mass = 5ul * 1ug/ul<!-- | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Probe''' | | align="center" style="background:#f0f0f0;"|'''Probe''' |
Revision as of 18:04, 16 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
- 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