Protocol

Preparation of Genomic DNA from Hawaiian Bobtail Squid (Euprymna scolopes) Tissue by Cesium Chloride Gradient Centrifugation

¹ Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA
² Kewalo Marine Laboratory, Pacific Biosciences Research Center/University of Hawaii at Manoa, Honolulu, HI 96813, USA
³ Department of Medical Microbiology, University of Wisconsin-Madison, Madison, WI 53706-1521, USA
⁴ Laboratory of Developmental Genetics, K.U. Leuven and VIB, B-3000 Leuven, Belgium

⁵Corresponding author (couet{at}hawaii.edu).

INTRODUCTION

This procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic methods and commercial kits for the preparation of genomic DNA based on proteolytic digestion of chromatin components, followed by selective binding of nucleic acids to ion-exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also, molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain conditions. Although nucleic acids prepared this way can serve as a template for polymerase chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these contaminants. The method described here yields high-molecular-weight DNA that can be readily restricted for Southern hybridization. The procedure uses brain tissue under the assumption that its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and avoids potential sources of enzymatic contaminants and parasites from the intestinal sac. However, the method can be applied to other tissue sources and works well with other species. The purification of DNA by gradient centrifugation is an established method based on the specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA, which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved and expensive and produces large amounts of ethidium bromide waste.