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Cosmid library of the marine macroalga Bryopsis maxima (Bryopsidales, Ulvophyceae)
Author(s) -
Inokuchi Ritsuko,
Motojima Kiyoto,
Okada Mitsumasa
Publication year - 2002
Publication title -
phycological research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.438
H-Index - 44
eISSN - 1440-1835
pISSN - 1322-0829
DOI - 10.1046/j.1440-1835.2002.00275.x
Subject(s) - cosmid , biology , microbiology and biotechnology , genomic library , dna , biochemistry , base sequence
SUMMARY The purpose of this study was to construct a cosmid library from chromosomal DNA of a marine macroalga, Bryopsis maxima Okamura ex Segawa (Bryopsidales, Ulvophyceae), in a rapid, simple and inexpensive manner. In the DNA purification, polysaccharides were removed by covalently binding them to resin particles containing free boric acid groups. The DNA yield was 20 μg g −1 of B. maxima fresh weight. This DNA was 100–200 kb in length, and its A 260 /A 280 and A 230 /A 260 ratios were 1.8 and 0.4, respectively. It was of sufficient quality for molecular research. The cloning procedures were carried out in the following steps: controlled partial shearing of purified DNA through a microsyringe, optimal size separation of the DNA by biased sinusoidal field gel electrophoresis, ligation of the DNA to the cosmid vector in the gel, and in vitro packaging into the lambda phage. The library consisted of 2.0 × 10 3 independent clones with an average insert size of 40 kb. The fragment amplified by polymerase chain reaction in the library was hybridized with a DNA fragment (328 bp) encoding B. maxima glutamate dehydrogenase under high‐stringency conditions by Southern blot analysis, thus demonstrating that the library contained B. maxima chromosomal DNA. This cosmid library is the first to be constructed for any species of marine macroalgae.