Abstract
A simple and cheap method of plasmid DNA preparation from phytopathogenic Gram-negative bacteria (Xanthomonad, Erwinia stewartii) is presented here. In this method, in place of the high-priced chemicals and commercial kits, available and cheap chemicals were used for rapid isolation of small and large plasmids from different Gram negative bacteria. The time also was reduced by using this method giving a high quality plasmid production as demonstrated on the agarose gel, which make this method be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A down scaled- protocol is also very useful for rapidly screening the wild plasmids in a large numbers of bacterial isolates in the experiments where hundreds of colonies should be screened for their plasmid contents such as in studying plasmid curing; antibiotic and heavy metal resistant bacteria or; xenobiotic compound degrading bacteria. Key words: Plasmid, DNA, Erwinia, Xanthomonas, curing.
Highlights
The preparation of plasmid DNA is one of most used techniques in the field of molecular biology
Different Gram negative bacterial strains belonging to Xanthomonas campestris pv. malvacearum (Xcm) and Erwinia stewartii were used in this study and obtained as lyophilized samples from the GSPB bacterial collection (Göttinger Sammlung phytopathologener Bakterien) (Table 1)
The earliest plasmid isolation procedures depended on the separation of covalently closed circles of plasmid DNA from chromosomal DNA fragment by ultra centrifugations on cesium chloride gradients containing high concentrations of ethidium bromide (Currier and Nester, 1976)
Summary
A simple and cheap method of plasmid DNA preparation from phytopathogenic Gram-negative bacteria (Xanthomonad, Erwinia stewartii) is presented here. In this method, in place of the high-priced chemicals and commercial kits, available and cheap chemicals were used for rapid isolation of small and large plasmids from different Gram negative bacteria. The time was reduced by using this method giving a high quality plasmid production as demonstrated on the agarose gel, which make this method be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments.
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