Kun:LabNotes/HiResChrPaint/2012-7-12: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Dye'''
| align="center" style="background:#f0f0f0;"|'''Dye'''
| align="center" style="background:#f0f0f0;"|'''Concentration(pmole/ul)'''
| align="center" style="background:#f0f0f0;"|'''Concentration(pmole/ul)'''
| align="center" style="background:#f0f0f0;"|'''Pooled probe conc(ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Labeling efficiency'''
| align="center" style="background:#f0f0f0;"|'''# fluorophores per probe'''
|-
|-
| #1-V4||278.49||0.70||Alexa-546||7.2
| #1-V4||278.49||0.70||Alexa-546||7.2||177||7.28%||5.8
|-
|-
| #2-V4||432.8||1.08||Alexa-488||24.3
| #2-V4||432.8||1.08||Alexa-488||24.3||177||15.81%||12.6
|-
|-
| #3-V4||359.76||0.90||Alexa-594||12
| #3-V4||359.76||0.90||Alexa-594||12||177||9.39%||7.5
|-
|-
| #4-V4||438.99||1.10||Alexa-647||2.0
| #4-V4||438.99||1.10||Alexa-647||2||177||1.28%||1.0
|-
|-
| #1-V6||425.3||1.06||Alexa-546||7.1
| #1-V6||425.3||1.06||Alexa-546||7.1||179||4.88%||3.9
|-
|-
| #2-V6||427.85||1.07||Alexa-488||18.4
| #2-V6||427.85||1.07||Alexa-488||18.4||179||12.58%||10.1
|-
|-
| #3-V6||426.04||1.07||Alexa-594||8.6
| #3-V6||426.04||1.07||Alexa-594||8.6||179||5.90%||4.7
|-
|-
| #4-V6||306.88||0.77||Alexa-647||2.0
| #4-V6||306.88||0.77||Alexa-647||2||179||1.91%||1.5
|-
|-
|  
|  

Revision as of 00:29, 13 July 2012

Dye coupling

  • Noi did the amplification and prepared single-stranded probes:Noi's lab note.
  • 1mL of nuclease free water was added to the Labeling Buffer tube containing sodium bicarbonate. The tube was placed on the Eppendorf thermal shaker at 37C for ~20 min.
  • Each vial of dye was resuspended in 2ul DMSO.
  • I took 2.5ul of DNA, add 1.5ul of Labeling buffer, then 1ul of dyes. I wasn't able to pipette the exact volume of the dyes due to inexperience and pipetting error.
Probe IDs Conc. (ng/ul) Total amount in 2.5ul (ug) Dye Concentration(pmole/ul) Pooled probe conc(ng/ul) Labeling efficiency # fluorophores per probe
#1-V4 278.49 0.70 Alexa-546 7.2 177 7.28% 5.8
#2-V4 432.8 1.08 Alexa-488 24.3 177 15.81% 12.6
#3-V4 359.76 0.90 Alexa-594 12 177 9.39% 7.5
#4-V4 438.99 1.10 Alexa-647 2 177 1.28% 1.0
#1-V6 425.3 1.06 Alexa-546 7.1 179 4.88% 3.9
#2-V6 427.85 1.07 Alexa-488 18.4 179 12.58% 10.1
#3-V6 426.04 1.07 Alexa-594 8.6 179 5.90% 4.7
#4-V6 306.88 0.77 Alexa-647 2 179 1.91% 1.5
  • The reaction mixes were incubated at room temperature in dark for 1 hour.
  • Add 5ul 3M NaOAC and 40ul H2O to each tube.
  • The labeled probes were purified individually with Zymo DNA Concentrator-5 columns.
    • Add 350ul of binding buffer, load the mixture to the column, spin at top speed for 30sec
    • Wash twice with 200ul Wash Buffer
    • Wash one additional time with 100ul 75% EtOH.
    • Eluted with 6ul H2O.
  • Four subsets of V4 probes were pooled in one tube, four other subsets for V6 probes were pooled in another tube.
  • Measure the yields with Nanodrop (fluorophore-specific concentrations are reported in the table above).
  DNA concentration (ng/ul)
  Chr.Paint.V4:   177ng/ul
  Chr.Paint.V6:   179ng/ul