Kun:LabNotes/HiResChrPaint/2012-7-12

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Dye coupling[edit]

  • 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)    Amount in 20ul
  Chr.Paint.V4:   177ng/ul        134 pmole
  Chr.Paint.V6:   179ng/ul        135 pmole
  • Diluted into 2pmole/ul for Chr.Paint.V6
20ul (original tube) + 47.5ul 1XTE = 67.5ul 
ship 20ul to Brian in Harvard
keep 20ul for Scripps
Store the rest at 4C (Rui, 4C box)