LAB2.NEW - Recombinant DNA Session 2: Plasmid Minipreps...

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Recombinant DNA Session 2: Plasmid Minipreps BACKGROUND The miniprep method , as the name implies, is a rapid, small scale method of obtaining plasmid DNA plus any insert DNA from bacterial cells. Absolute purity is not obtained and usually not essential. Two general variations of miniprep method are available - the boiling method and the alkaline lysis method . You will learn how to do the alkaline lysis method. In this method, the bacterial cells containing plasmid are first harvested from the culture broth and resuspended in a small volume. The cells are lysed by treating with a strong detergent (SDS) and a strong base (sodium hydroxide), denaturing cellular proteins at the same time E. coli protein is precipitated with the bulk of E. coli chromosomal DNA, leaving most plasmid DNA in the supernatant, contaminated by RNA and residual protein and DNA. Neutralization and precipitation are achieved by the addition of a high concentration of potassium acetate. After spinning out the precipitate, the supernatant containing plasmid DNA may or may not be extracted with a phenol/chloroform mixture to further deproteinize the plasmid DNA. The plasmid DNA is then alcohol precipitated itself by the addition of about two volumes of isopropanol. The precipitation accomplishes several aims: 1. It concentrates the sample 2. It helps remove contaminating salts. The conditions for alcohol precipitation used in a plasmid miniprep are different than used for most DNA precipitation. Room temperature precipitation, short waiting time, and room temperature centrifugation are done in a plasmid miniprep. The purpose is to minimize co-precipitation of contaminants. Plasmid DNA concentration is relatively
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This note was uploaded on 05/03/2008 for the course BIO 2322 taught by Professor Spotswood during the Spring '08 term at The University of Texas at San Antonio- San Antonio.

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LAB2.NEW - Recombinant DNA Session 2: Plasmid Minipreps...

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