• 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

        Fill-in Labeling of DNA : directional end label

        互联网

        1094

        http://axon.med.harvard.edu/~cepko/protocol/mike/F1.html

        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 m l) aliquotes at -80 degrees and thaw on ice just prior to use

        10 X Klenow Buffer

        0.5 M Tris 7.5 500

        0.1 M MgCl2

        m l 1 M Tris 7.5 2 100 m l 1 M MgCl2

        10 mM DTT 100

        0.5 mg/ml BSA 50

        250ml 0.1 M DTT m l 10 mg/ml BSA m l Q store at -20℃ in 50 m l aliquotes

        dNTP Mix

        21

        3

        Note: leave out the dNTP which will be used for labeling of the chosen restriction site (i.e.

        Procedure Digest 2

        Resuspend the pellet in 19

        25

        5

        5

        1

        Incubate at room temperature for 30'.

        Phenol/chloroform extract and EtOH ppt. Resuspend in 16

         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

        Count 1

        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.

        m l Q m l each of 3 cold dNTP's (10 mM stocks, see above) a - 32 P-dATP with EcoRI Labeling). m g 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. m l Q, then add: m l dNTP mix m l 10X Klenow Buffer m l a 32 P dNTP m l Klenow m l Q and digest with the second enzyme for 30' at 37 degrees. m l Q. m l 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.

        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