Please cite as: CSH Protocols; 2007; doi:10.1101/pdb.prot4891

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Micrococcal Nuclease-Southern Blot Assay: II. Capillary Transfer and Hybridization

Michael Carey and Stephen T. Smale

This protocol was adapted from "In Vivo Analysis of an Endogenous Control Region," Chapter 10, in Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, by Michael Carey and Stephen T. Smale. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.


INTRODUCTION

This is the second phase of a two-part assay for exploring whether an endogenous control region of interest is assembled into nucleosomes or is devoid of nucleosomes. This assay can also be used to determine if nucleosomes are similarly positioned at the locus in every cell within a population. In this second part of the assay, the restricted genomic DNA is separated by agarose gel electrophoresis and stained with ethidium bromide, resulting in a ladder of bands corresponding in size to multiples of the nucleosome core plus linker (~200 bp). To determine whether a DNA fragment of interest is nucleosomal, the genomic DNA is subjected to Southern blot analysis. If a probe derived from the DNA fragment hybridizes to the ladder of nucleosomal bands, the fragment may indeed be assembled into nucleosomes.


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