Matt:LabNotes/2015-7-22: Difference between revisions

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=Dye coupling SNAP25 odd&even 488&594=
=Dye coupling SNAP25 odd&even 488&594=
==Combine SNAP25_odd and SNAP25_even==
*Combined SNAP25 odd probes (24) and even probes (24) following same protocol as [[Matt:LabNotes/2015-7-8#Combining_CUX2_odd_and_CUX2_even | CUX2 odd&even]]
*Combined SNAP25 odd probes (24) and even probes (24) following same protocol as [[Matt:LabNotes/2015-7-8#Combining_CUX2_odd_and_CUX2_even | CUX2 odd&even]]
**SNAP25_odd: 1148.3 ng/ul
**SNAP25_odd: 1148.3 ng/ul
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#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 7.5 uL DNA (~1ug/ul) and 4.5 uL of sodium bicarbonate buffer
#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
#*Dye cannot be saved for later use.  Use immediately!
#*Dye cannot be saved for later use.  Use immediately!
#Add 8 uL sample to dye tube
#Add 8 uL sample to dye tube (Mixed 12ul but after heating only ~8ul left)
#Incubate in the dark for 1 hour
#Incubate in the dark for 1 hour
#Add 10 uL 3M NaOAc and 80 uL '''H2O''' to sample
#Add 10 uL 3M NaOAc and 80 uL '''H2O''' to sample
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#*No vacufuge
#*No vacufuge
#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)
#Remove supernatant and add 750 uL chilled 75% 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 = 7.5ul * 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;"|'''594 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_odd-488||413.9||66.1||-0.1||1.04||5.0||7.5||66.667
|-
| SNAP25_even-594||455.3||-1.2||89||1.27||5.5||7.5||73.333
|-
| SNAP25_even-594||477.3||-1.4||88.3||1.20||5.7||7.5||76
|}

Latest revision as of 21:41, 23 July 2015

Dye coupling SNAP25 odd&even 488&594[edit]

Combine SNAP25_odd and SNAP25_even[edit]

  • Combined SNAP25 odd probes (24) and even probes (24) following same protocol as CUX2 odd&even
    • SNAP25_odd: 1148.3 ng/ul
    • SNAP25_even: 1154.0 ng/ul

Dye coupling[edit]

Dan's best practice dye coupling protocol
Generic dye coupling protocol

  • SNAP25_odd-488
  • SNAP25_even-594
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 7.5 uL DNA (~1ug/ul) and 4.5 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. 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!
  5. Add 8 uL sample to dye tube (Mixed 12ul but after heating only ~8ul left)
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL H2O to sample
    • Previously did 80ul TE but maybe H2O will work better because no amine groups
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  10. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  11. Remove supernatant and add 750 uL chilled 75% EtOH
  12. Centrifuge 5 minutes at 4 C
  13. Dry the pellet in the hood
  14. Resuspend pellet in 12 uL TE
  15. 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 = 7.5ul * 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 (%)
SNAP25_odd-488 413.9 66.1 -0.1 1.04 5.0 7.5 66.667
SNAP25_even-594 455.3 -1.2 89 1.27 5.5 7.5 73.333
SNAP25_even-594 477.3 -1.4 88.3 1.20 5.7 7.5 76