Accublue

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Objective

A protocol for quantifying dsDNA using a dsDNA specific dye binding assay that is unaffected by common contaminants e.g. RNA or oligonucleotides.

Step by step instructions

Still a work in progress

    1. Prepare DNA standards starting with a stock solution of calf thymus DNA at 250 ng/µl.
    Tube Add µl of µl NFW Final conc
    A 50 stock 0 250
    B 37.5 stock 12.5 187.5
    C 25 stock 25 125
    D 25 stock 75 62.5
    E 25 D 25 31.25
    F 12.5 E 37.5 15.625
    G 6.25 F 43.75 7.8125
    H 0 na 75 0

    1. Prepare working dye dilution.
      • 1 µl dye, 1 µl enhancer, and 100 µl dilution buffer per sample or standard.
      • keep dye cold and dark.
    2. Set up the plate.
      • Add 100 µl diluted dye to each well
      • then add 2 µl each sample or standard
    3. Measure fluorescence.
      • Ex = 350, Em = 460
    4. Calculate concentrations.
      • require that R^2 > 0.95
      • and that your sample fluorescence is within the linear range of the curve

    History

    Created 18:15 Apr 07, 2016 By: EliMeyer

    Last updated 05:56 Jun 26, 2019 By: Admin