• 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

        De-phosphorylation of DNA

        互联网

        803

         

        De-phosphorylation of DNA

        The preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonucleases.

        You will need:

        10 x OPA+ Buffer (100mM Tris.acetate, pH 7.5, 100mM magnesium acetate, 500mM potassium acetate)
        Calf Intestinal Phosphatase (CIP, Promega)
        Sterile, nano-pure water

        1) After digestion of DNA with the appropriate restriction endonuclease, phenol/chloroform extract, precipitate and re-dissolve the DNA in, typically, 89ul of sterile, distilled H2 O.

        2) To this solution add 10ul of 10 x OPA+ and 1ul of a 0.1 unit/ul of CIP in 1 x OPA+.

        N.B: Do not use more that 0.1 units CIP if it can be avoided as inactivation of CIP by heat is not 100% effective if more than 1 unit is used.

        3) Incubate at 37°C for 30 minutes.

        4) After incubation is complete, heat inactivate the CIP by incubating the solution at 85°C for 15 minutes.

        5) Cool the reaction to room temperature, extract once with phenol:chloroform and once with chloroform.

        6) Precipitate DNA with ethanol and recover by centrifugation in a microfuge.

        7) Wash the pellet once in 80% ethanol, vacuum dry and resuspend in the appropriate buffer.

         

        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