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KMID : 0359320070470020157
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Korean Journal of Veterinary Research
2007 Volume.47 No. 2 p.157 ~ p.162
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Evaluation and modification of alkaline Lysis plasmid preparation method from Lactobacillus spp.
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Lee Deog-Yong
Seo Yeon-Soo
Kang Sang-Gyun
Yoo Han-Sang
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Abstract
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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℃. Only incubation at -70℃ 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.
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KEYWORD
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extraction yield, Lactic acid bacteria, LAB, plasmid DNA, purity
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