The EpiMark® Nucleosome Assembly Kit is used to make unmodified recombinant human nucleosomes with user-supplied DNA or the provided control DNA. Purified recombinant human Histone H2A/H2B Dimer and Histone H3.1/H4 Tetramer are mixed with DNA at 2 M NaCl. To generate nucleosomes, the salt concentration is lowered by dilution or dialysis allowing each histone tetramer to associate with two histone dimers and form the histone octamer on the DNA (1,2). A method for assaying nucleosome formation by gel shift assay is included. These nucleosomes may serve as better substrates for enzymes that are inactive on the DNA or one of the core histones alone (3,4). Each reaction creates nucleosomes from ~1 µg of 208 bp DNA and may be scaled depending on the experiment.
Nucleosome Assembly
A gel shift assay allows visualization of complex formation when various ratios of Octamer* to DNA are used to form mononucleosomes using the provided nucleosome control DNA. When assembly reactions are run on 6% polyacrylamide gel in 0.5X TBE, a characteristic shift occurs from 208 bp to approximately 700 bp.
M: Low Molecular Weight DNA Ladder (NEB #N3233)
Lane 1: Nucleosome Control DNA
Lane 2: 0.5:1 ratio of Octamer* to DNA
Lane 3: 1:1 ratio of Octamer* to DNA.
*Octamer = 2:1 mix of Histone H2A/H2B Dimer and Histone H3.1/H4 Tetramer.
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Trademarks
EPIMARK® is a registered trademark of New England Biolabs, Inc. TRIDYE™ is a trademark of New England Biolabs, Inc.
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