Matt:JustinLabNotes/2015-7-20: Difference between revisions
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>Mzcai (Created page with "==Dye Coupling== SNAP25-488 241 41.3 0 37.1 1.11 57.8 2.892 CUX2-647 245.3 0.4 42.1 37.8 1.11 58.9 2.9436") |
>Mzcai mNo edit summary |
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=Dye-Coupling with Alexa 488 and Alexa 647= | |||
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> | |||
[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] | |||
SNAP25-488 241 41.3 0 | SNAP25-488 | ||
CUX2-647 245.3 0.4 42.1 | CUX2-647 | ||
#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 SNAP25 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 | |||
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation | |||
#*Use 1X TE Buffer | |||
#*No vacufuge | |||
#*'''USED 2 COLUMNS FOR CUX2-647, HALF VOLUME IN EACH | |||
#**Test whether decreasing loaded amount might more efficiently remove free dyes | |||
#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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''647 dye (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' | |||
|- | |||
| SNAP25-488||241||41.3||0||1.11||2.89||5||57.8 | |||
|- | |||
| CUX2-647||245.3||0.4||42.1||1.11||2.94||5||58.9 | |||
|} |
Latest revision as of 18:24, 21 July 2015
Dye-Coupling with Alexa 488 and Alexa 647[edit]
Dan's best practice dye coupling protocol
Generic dye coupling protocol
SNAP25-488 CUX2-647
- 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 SNAP25 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
- USED 2 COLUMNS FOR CUX2-647, HALF VOLUME IN EACH
- Test whether decreasing loaded amount might more efficiently remove free dyes
- 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) | 647 dye (pmol/ul) | dye:probe ratio | ssDNA mass (ug) | ssDNA start mass (ug) | ssDNA Yield (%) |
SNAP25-488 | 241 | 41.3 | 0 | 1.11 | 2.89 | 5 | 57.8 |
CUX2-647 | 245.3 | 0.4 | 42.1 | 1.11 | 2.94 | 5 | 58.9 |