分离假单胞菌大质粒的简便方法

本文介绍了一种分离假单胞菌大质粒的简便方法。用这种方法提取的质粒DNA样品,可直接用于琼脂糖凝胶电泳检查和限制性内切酶分析。

A SIMPLE METHOD FOR THE ISOLATION OF LARGE PLASMIDS IN PSEUDOMONAS

A simple method is described for the isolation of large plasmids in Pseudomonas. A 4ml sample of a culture growing in nutrient broth to an optical density at 600nm of 1.0 was centrifuged. The cell pellet was suspended in 1 ml of TE buffer (50 mM Tris, 20mM Na₂EDTA, pH 8.0) and lysed with 2 ml of lysing solution (3 % SDS, 0.2N NaOH). After the solution had been left on ice for 10 min, 1 ml of TS solution (lM Tris, 4M NaCl, pH 7.0) was added and the mixture was left on ice for another 30min. The suspension was centrifuged, and the supernatant was mixed with an equal volume of ethanol. The mixture was incubated on ice for one hour and then centrifuged. The pellet was dissolved in 0.1ml of TES solution (50mM Tris, 5mM Na₂EDTA, 50mM NaCl, pH 8.0) and extracted with 0.1ml of chloroform. The aqueous solution containing plasmid DNA can be used directly for agarose gel electropho-resis and restrictive endonuclease analysis.