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  • A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis.

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis.

Nature protocols (2007-04-05)
Kaichun Zhu, Huali Jin, Zhonghuai He, Qinghong Zhu, Bin Wang
초록

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.

MATERIALS
제품 번호
브랜드
제품 설명

Sigma-Aldrich
Ethidium bromide, BioReagent, for molecular biology, powder
Sigma-Aldrich
Trizma® hydrochloride solution, pH 7.0, BioPerformance Certified, 1 M, suitable for cell culture