Korean Journal of Veterinary Research 2007;47(2):157-162.
Evaluation and modification of alkaline lysis plasmid preparation method from Lactobacillus spp.
Deog-Yong Lee, Yeon-Soo Seo, Sang-Gyun Kang, Han Sang Yoo
College of Veterinary Medicine, KRF Zoonotic Disease Priority Research Institute and Brain Korea 21 Program for Veterinary Science, Seoul National University
Abstract
Lactic acid bacteria (LAB) has been regarded as a useful microorganism and tried to manipulate plasmid DNA for increasing the usefulness. Although several methods have been developed to isolate plasmid DNA from Escherichia coli (E. coli), these methods were not sufficient to apply to LAB with exception of O'Sullivan's lysis method. So, we evaluated plasmid DNA extraction from LAB using general E. coli preparation methods and tried to improve the extraction yield and DNA purity by modifying O'Sullivan's alkaline lysis method. To improve the extraction yield, salt and carrier were added to precipitant and those were incubated at $-70{^{circ}C}$. Only incubation at $-70{^{circ}C}$ was the effective method of those modifications. Purity of plasmid DNA was improved by two times of each centrifugation and phenol/chloroform extraction. However, DNA was damaged by twice extraction with phenol/chloroform. Also, exclusion of ethidium bromide showed negative effect to purity. Additionally, it was recommended that improvement of the extraction yield may be due to centrifugation at high speed for more time and to dissolving complete DNA pellet before addition of 7.5 M ammonium acetate. Extraction using this modification produced higher quality of plasmid DNA.
Key Words: extraction yield, Lactic acid bacteria (LAB), plasmid DNA, purity
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