• 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

        Transformation DNA fragments (or plasmid DNA) into competent E. coli

        互联网

        1697

        Transformation DNA fragments (or plasmid DNA) into competent E. coli

        1.Remove competent cells (E.coli DH5aTM from GIBCO BRL) from -70 ℃ freezer; thaw on wet ice.

        2.Place four 15-ml modified polystylene tubes (PST; Corning disposable sterile centrifuge tube) on ice.

        3.Gently mix cells (tapping with fingers), then aliquot 50ʵl competent cells into each of chilled the 15 ml PST.

        4.Add 1 µl of recombinant DNA sample (1 - 2 µg DNA) to the competent cells by moving the pipette through the cells while dispensing. Gently tap tubes to mix.

        5.Incubate cells on ice for 30 min.

        6.Heat-shock cells 45 sec in a 42 ℃ water bath: Do NOT shake.

        7.Place on ice for 2 min

        8.Add 0.95 ml of room temperature SOC.

        9.Add 5, 10, 20, 50, 100, and 200 µl (duplicate) of the diluted DNA sample into 2.5 ml of 0.8 % LB at 42℃. Do this step and the next step within 5 min (-42℃).

        10.Do overlay on LB containng Carbenicillin plates (filter sterilized Crb 100 µg/ml or Amp 100 µg//ml).

        11.Incubate at 37℃.

        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