• My NEB
  • Print
  • PDF
  • Gel Shift Assay for EpiMark™ Nucleosome Assembly Kit (E5350)

    Protocol

    1. Mix 10 µl of each sample with 2 µl of 100% glycerol, including control reactions.

    2. Load 10 µl of each sample onto a 6% DNA retardation gel. We recommend running 2 µl TriDye™ 100 bp DNA Ladder (NEB# N3271) alongside reactions.

    3. Electrophorese according to gel manufacturer protocol in buffer containing no ethidium bromide. Using the marker lane to track the gel run, run the blue dye off the gel and the green dye about two thirds down the gel. Xylene Cyanol in these gels runs approximately where the 200 bp substrate runs.

    4. Soak gel for 3 to 5 minutes in 1X TBE with ethidium bromide.

    5. Analyze gel.
      • Should see a gel shift of the 208 bp band when bound by octamer to about 700 bp.
      • Free DNA stains much better by ethidium bromide than nucleosome bound complex.
      • Optimal concentration is the lowest concentration of octamer needed to see most of the DNA shifted with the least amount of higher order aggregates.
      • If not in the range, then repeat experiment increasing or decreasing the amount of octamer used until an optimal ratio is found.