Authors
Kayoko Hayashihara, Susumu Uchiyama, Shouhei Kobayashi, Masanobu Yanagisawa, Sachihiro Matsunaga, and Kiichi Fukui
Summary
Given that DNA on which genomic information is written exists as chromosomes in a cell, handling chromosomes in vitro as experimental materials can provide varieties of information throughout life sciences. Metaphase chromosomes are highly delicate under in vitro conditions, moreover, it has been difficult to prepare massive chromosomes as experimental materials. These inconvenient points have prevented researchers to use chromosomes as the materials for in vitro experiments, although numerous microscopic observations have been so far performed. There is a standard protocol to prepare mitotic metaphase chromosomes, i.e., PA method1, 2, 5-7. However the chromosomes prepared by the method have been found to contain lots of contaminated proteins4. Ordinary purification processes, i.e., the sucrose density gradient centrifugation4 often or usually result in tremendous decrease in chromosome yield. Thus the purity and the quantity have never met in chromosome sample preparation. Recently we have developed a new method based on several previously published protocols1-4. The method enables obtainment of intact and highly purified chromosomes in large quantities. The protocol consists of two steps; isolation of chromosomes from the synchronized mitotic human cells by the improved PA procedure, and purification of the chromosomes by Percoll density gradient centrifugation (PDGC).Further details
The protocol was published on Protocol Exchange in 2008. To see the entire protocol, click on the source link.Advertisement
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