• 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

        DNA Isolation from Agarose Gels with DEAE Paper

        互联网

        831
               

        MATERIALS:

        The DEAE paper can be used as supplied by Schleicher and Schuell, however the binding capacity can be increased by washing the paper in 10mM EDTA, pH 7.5 for 10 minutes, 0.5M NaOH for 5 minutes, followed by several rinses in distilled water. The paper can be stored at 4o C for several months.

        NET buffer:

        0.15M NaCl
        0.1mM EDTA
        20mM Tris-HCl, pH 8.0

        High salt NET buffer:

        1.0M NaCl
        0.1mM EDTA
        20mM Tris-HCl, pH 8.0

        METHOD:

        After the DNA bands have been separated electrophoretically (at around 80 V),the gel is viewed under long wave UV light and a slit cut in the gel,just ahead of the desired band.A piece of DEAE paper is inserted in the slit.Another piece of DEAE paper can be inserted behind the desired band,to prevent unwanted bands being trapped by the paper.

        Electrophoresis is continued for approximately 10 to 15 minutes with the voltage increased to 100-120 V.

        The DEAE paper is removed and binding of the DNA checked by viewing the paper with long wave UV light.The paper is then placed in an eppendorf tube containing 500 μl  NET buffer to wash away remaining agarose.The bound DNA can now be stored at 4℃ for several days.The DNA should always remain covered with buffer to prevent irreversible binding which may occur if the paper is allowed to dry out.

        To elute the bound DNA ,place the DEAE paper in an eppendorf tube containing 250 μl  of high salt NET buffer so that the paper is completely covered.Make sure that the side of the paper containing the DNA faces the buffer and not the side of the tube.If necessary,centrifuge briefly to submerge the paper.

        Incubate the tube at 55-68℃ for 45 minutes with occassional swirling (or until all the DNA has been eluted as checked under long wave UV light).

        Remove the buffer to a fresh tube and add 50 μl high salt NET buffer to wash the membrane.Remove the additional buffer and add to the fresh tube.

        Centrifuge the buffer for 30 seconds to pellet any remaining DEAE paper fibres and remove the buffer to a fresh tube (leaving approx.20 μl  in the bottom of the tube).

        Extract the residual ethidium bromide with 3 volumes of water saturated butanol.

        Remove and retain the bottomlayer and add 1/20 volume of 3.0M sodium acetate,pH 5.25 and 2.5 volumes of cold absolute ethanol.Incubate at -70℃ for at least one hour.Ethanol precipitate and vacuum dry.

        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