Daniel:Notebook/PosSequencing/2017-4-27

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Emulsion PCR (Started Yesterday)[edit]

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Protocol Part 3-Hybridization and Imaging[edit]

Probe hybridization and washes based on Matt's experiment.

Calculations for dye binding:

50 × 10-3 mg beads × 500pmolmg = 25 pmol available template @ max


  1. Fluorescent Oligo Binding
    1. Put suspension on magnet
    2. Resuspend beads in 25 uL 4X SSC buffer
    3. Make the following dye hybridization buffer
      1. 2 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
      2. 15 uL formamide
      3. 4 uL nfH2O
    4. Add 25 uL fluorescent probe in 60% formamide
    5. Incubate at room temp in the dark for 30 min
    6. Wash three times in 2X SSC
    7. Resuspend in 50 uL 2XSSC
  2. Imaging