Matt:LabNotes/2015-7-14: Difference between revisions
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#*No vacufuge | #*No vacufuge | ||
#Repeat column purification | #Repeat column purification | ||
#*[[File:IMG_0526.JPG |450px]] | |||
#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) | ||
Line 27: | Line 28: | ||
#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''' | ||
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| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' | | align="center" style="background:#f0f0f0;"|'''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 | ||
|- | |- | ||
|} | |} | ||
Revision as of 19:46, 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
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 |