Matt:JustinLabNotes/2015-7-20: Difference between revisions

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==Dye Coupling==
=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 37.1 1.11 57.8 2.892
SNAP25-488
CUX2-647 245.3 0.4 42.1 37.8 1.11 58.9 2.9436
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

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
  3. Make 2 25ul aliquots of SNAP25 48probe to prevent so many freeze-thaws in the future
  4. Denature samples for 5 minutes at 95C, then snap cool using ice box
  5. 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!
  6. Add 8 uL sample to dye tube
  7. Incubate in the dark for 1 hour
  8. Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
  9. 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
  10. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  11. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  12. Remove supernatant and add 750 uL chilled 80% EtOH
  13. Centrifuge 5 minutes at 4 C
  14. Dry the pellet in the hood
  15. Resuspend pellet in 12 uL TE
  16. 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