Technical Note
Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen
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* Corresponding authors: Michael J Buck mjbuck@buffalo.edu - Joel A Huberman huberman@buffalo.edu
1 Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
2 Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
3 Department of Biochemistry and Center of Excellence in Bioinformatics and Life Sciences, SUNY at Buffalo, 701 Ellicott St., Buffalo, NY 14203, USA
BMC Research Notes 2011, 4:499 doi:10.1186/1756-0500-4-499
Published: 16 November 2011Abstract
Background
Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.
Findings
Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.
Conclusions
We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.