• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Miniprep/Kit-free high-throughput protocol

        互联网

        771

         

        Background

        This protocol is adapted from "Molecular Cloning: A Laboratory Manual", Second Edition, Sambrook, Fritsch, and Maniatis. It is a quick, inexpensive way to purify large numbers of plasmids (I used to routinely do 80 at a time.--Kathleen) and yields DNA that is clean enough for sequencing or for use as a PCR template.

        Protocol

        1. Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells.
        2. Remove and discard the supernatent.
        3. Add 300 μL STET buffer, and resuspend cells by vortexing.
        4. Add 10 μL lysozyme (10 mg/mL), vortex, and submerse in boiling water for 40 sec.
        5. Spin for 30 min in a tabletop centrifuge at maximum speed at 4 ˚C.
        6. Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don''t get any cellular debris on the sides of the tube.
        7. Add 300 μL ice cold isopropanol to precipitate the DNA (or 300μL of 2:1 isopropanol:ammonium acetate, mixed just before you use it. See this discussion of precipitating nucleic acids.)
        8. Spin for 10 min in a tabletop centrifuge at maximum speed at 4 ˚C.
        9. Remove supernatent.
        10. Add 200 μL ice cold 80% ethanol to wash pellet and spin for 5 min in a tabletop centrifuge at maximum speed at 4 ˚C.
        11. Remove supernatent.
        12. Dry pellet (air dry at room temperature or 37 ˚C or dry in a speedvac).
        13. Rehydrate in 50 μL TE.

        Buffers

        STET

        8% sucrose
        50 mM Tris-HCl, pH 8
        0.5% Triton X-100
        50 mM EDTA

        TE

        10 mM Tris-HCl, pH 8
        1 mM EDTA

         

        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序