• 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

        One-step Transformation of E. coli

        互联网

        1058

        TRANSFORMATION (ONE-STEP PEG METHOD)

        1.Dilute 1:100 a fresh O/N culture of bacteria into prewarmed LB broth and incubate cells with shaking (225 rpm)to an OD600 of 0.3-0.4.

        2.Add an equal volume of ice-cold 2X TSS and mix gently.[TSS is LB broth with 10% PEG (MW3350-8000),5% DMSO,and 20-50 mM Mg2+ (MgSO4 or MgCl2)at a final pH of 6.5].

        3.For long-term storage,cells are frozen immediately in a dry ice/ethanol bath and stored at -70℃.

        For transformation,a 0.1 ml aliquot of cells is pipetted into a cold polypropylene tube containing 1μl (100 pg)od plasmid DNA,and cell/DNA suspension is mixed gently.[When frozen cells are used,cells are thawed slowly on ice and used immediately.]

        4.The cell/DNA mixture is incubated for 5-60 min at 4℃.

        5.A 0.9 ml aliquot of TSS (or LB broth)plus 20 mM glucose is added,and cells are incubated at 37℃ with shaking (225 rpm)for 1 hour to allow expression of the antibiotic-resistance gene.

        6.Transformants are selected by standard methods.

        PNAS (1989)86:2172-2175.CT Chung,SL Niemela,RH Miller.

        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