Kun:LabNotes/HiResChrPaint/2012-7-12

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Dye coupling

  • Noi did the amplification and prepared single-stranded probes.
  • 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
#1-V4 278.49 0.70 Alexa-546
#2-V4 432.8 1.08 Alexa-488
#3-V4 359.76 0.90 Alexa-594
#4-V4 438.99 1.10 Alexa-647
#1-V6 425.3 1.06 Alexa-546
#2-V6 427.85 1.07 Alexa-488
#3-V6 426.04 1.07 Alexa-594
#4-V6 306.88 0.77 Alexa-647
  • The reaction mixes were incubated at room temperature in dark for 1 hour.
  • The labeled probes were purified individually with Zymo DNA Concentrator-5 columns.
  • Four subsets of V4 probes were pooled in one tube, four other subsets for V6 probes were pooled in another tube.