• 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片段的Fill-in标记

        互联网

        1903
         

        This protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA probes.

        Solutions

        10 mM dNTP Stocks

        Thaw100 mM stocks (NEB or Boehringer Mannheim) on ice and dilute 10-fold in Q.

        store small (10-20 ml) aliquotes at -80 degrees and thaw on ice just prior to use

         

        10 X Klenow Buffer

        0.5 M Tris 7.5 500 ml 1 M Tris 7.5

        0.1 M MgCl2 100 ml 1 M MgCl2

        10 mM DTT 100 ml 0.1 M DTT

        0.5 mg/ml BSA 50 ml 10 mg/ml BSA

        250 ml Q

        store at -20° in 50 ml aliquotes

         

        dNTP Mix

        21 ml Q

        3 ml each of 3 cold dNTP's (10 mM stocks, see above)

        Note: leave out the dNTP which will be used for labeling of the chosen restriction site (i.e. a-32P-dATP with EcoRI Labeling).

        Procedure

        • Digest 2 mg of CsCl purified plasmid DNA (Protocol C.1) with the restriction endonuclease corresponding to the end to be labeled. phenol/chloroform extract and EtOH ppt.

        • Resuspend the pellet in 19 ml Q, then add:

        25 ml dNTP mix

        5 ml 10X Klenow Buffer

        5 ml a 32P dNTP

        1 ml Klenow

        Incubate at room temperature for 30'.

        • Phenol/chloroform extract and EtOH ppt. Resuspend in 16 ml Q and digest with the second enzyme for 30' at 37 degrees.

        • Gel purify by running out the restriction digest (5 minutes 100 mA) on a 1% minigel and spin purifying (Protocol D.5).

        • Resuspend the purified fragment in 100 ml Q.

        • Count 1 ml by spotting onto Whattman 3mm filter paper and counting for 1 min. I get 20,000-50,000 cpm for restriction fragments and 200,000-400,000 cpm for double stranded oligos. Anything less than 15,000 for restriction fragments should be trashed.

        • Probes labeled using this protocol are usually good for 1-2 weeks but the best results are obtained when the probe is used within the first few days after labeling.

        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