• 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

        Plasmid DNA Isolation from Bacteria

        互联网

        1219

         Materials:

        TE buffer

        10% (w/v) sodium dodecyl sulfate (SDS)

        20 mg/ml proteinase K

        phenol\chloroform (50:50)

        isopropanol

        70% ethanol

        3M sodium acetate pH 5.2

        Phase Lock GelTM (5 Prime, 3 Prime, Inc)

        1.Grow E. coli culture overnight in rich broth. Transfer 1.5ml to a micro centrifuge tube and spin 2min. Decant the supernatant. Repeat with another 1.5ml of cells. Drain well onto a Kimwipe.

        2.Resuspend the pellet in 467μl TE buffer by repeated pipetting. Add 30μl of 10% SDS and 3μl of 20mg/ml proteinase K, mix , and incubate 1 hr at 37EC.

        3.Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed. CAUTION: PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB COAT AND KEEP TUBES CAPPED TIGHTLY. Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin 2min.

        4.Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a new Phase Lock GelTM tube and spin 5 min. Transfer the upper aqueous phase to a new tube.

        5.Add 1/10 volume of sodium acetate. Mix.

        6.Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates.

        7. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end).

        8. Wash DNA by dipping end of rod into 1ml of 70% ethanol for 30 sec.

        9. Resuspend DNA in 100-200μl TE buffer. Complete resuspension may take several days.

        10. Store DNA at 4EC short term, -20 or -80EC long term

        11. After DNA has dissolved, determing the concentration by measuring the absorbance at 260nm.

        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