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

Info icon This preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
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.
Image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern