Accuclear

From Wiki | Meyer Lab
Jump to: navigation, search

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 96 stock 304 60
    B 72 stock 328 45
    C 48 stock 352 30
    D 100 A 300 15
    E 71 B 329 8
    F 53 C 347 4
    G 53 D 347 2
    H 0 na 400 0

    1. Prepare working dye dilution.
      • 1 µl dye 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 = 468, Em = 507
    4. Calculate concentrations.
      • require that R^2 > 0.95
      • and that your sample fluorescence is within the linear range of the curve

    History

    Created 20:47 Mar 12, 2016 By: EliMeyer

    Last updated 08:50 Sep 23, 2019 By: Admin