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Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis
Author(s) -
LEWINGTON J.,
GREENAWAY S. D.,
SPILLANE B. J.
Publication year - 1987
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.1987.tb01612.x
Subject(s) - genomic dna , pseudomonas putida , biology , dna , restriction enzyme , plasmid , in vitro recombination , lysis , molecular cloning , plasmid preparation , multiple cloning site , cloning (programming) , microbiology and biotechnology , complementary dna , genetics , bacteria , recombinant dna , gene , pbr322 , vector (molecular biology) , computer science , programming language
We describe a rapid procedure for extracting purified chromosomal DNA from Pseudomonas putida , and some minor modifications needed for use in other organisms, including Gram‐positive strains. The technique is rapid and generally produces between 1 and 5 μg of DNA from 1 ml of liquid culture. The DNA is highly purified and can be readily cut with small quantities of restriction endo‐nucleases, cloned into plasmid vectors and used as a substrate for hybridization with labelled DNA probes. Genetical analysis and cloning of genomic DNA from environmentally important organisms like Ps. putida has become increasingly important. Bacterial chromosomal DNA is usually prepared from large volumes of liquid culture, and involves time‐consuming steps necessary to purify the DNA sufficiently for use in cloning experiments (e. g. Marmur 1961). We have developed a method for preparing small quantities of genomic DNA, which involves whole cell lysis and purification by precipitation and centrifugation. Loss of DNA occurs by shearing, but the yield of DNA is sufficient, and the quality is high. The method has been used to prepare DNA from a variety of organisms and is particularly applicable where the preparation and analysis of DNA from a large number of isolates is required.