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    Miniprep of Plasmid DNA (Alkaline Lysis Method)

    互联网

    646

    实验步骤

     

    1. Inoculate 2 ml of L2 media containing the appropriate antibiotic with a single bacterial colony picked using a sterile toothpick. Incubate at 37 degrees overnight with vigorous shaking.

    2. Transfer 1.5 ml of culture into an Eppendorf tube. Spin for 30 seconds to pellet cells.

    3. Remove the medium by pipet or aspiration, leaving the pellet as dry as possible.

    4. Resuspend pellet in 100 ul of ice-cold GTE solution by gently pipetting up and down. Let stand 5 minutes at room temperature. Be sure to completely resuspend cells. Avoid creating bubbles.

    5. Add 200 ul of freshly prepared NaOH/SDS solution and mix by inverting tube rapidly or by tapping. Do not vortex. Let stand on ice 5 minutes. This treatment lyses the bacteria. SDS denatures bacterial proteins and NaOH denatures chromosomal and plasmid DNA.

    6. Add 150 ul of KOAc solution and vortex to mix. Let stand on ice for 5 minutes. This neutralizes the mixture, causing the plasmid DNA to reanneal rapidly. Most of the chromosomal DNA and bacterial proteins precipitate along with the SDS-potassium complex.

    7. Centrifuge for 5 minutes at 4 degrees.

    8. Transfer the supernatant to a fresh Eppendorf tube.

    9. Add an equal volume of Phenol/chloroform and mix by vortexing. Centrifuge for 2 minutes at room temperature and transfer the upper, aqueous layer to a fresh Eppendorf tube.

    10. Add 2 volumes of ethanol and let stand at room temperature for 2 minutes.

    11. Centrifuge for 5 minutes at room temperature. The prior three steps further purify and concentrate the plasmid DNA.

    12. Aspirate off the supernatant and dry completely in a Speed-Vac concentrator. Resuspend pellet in 30 ul of TE w/RNase.

    Note: 3 ul of DNA is an appropriate volume to use in a typical analytical restriction digest reaction.

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