• 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

        Coating complexes with pHPMA

        互联网

        925

         

        1. DNA is dissolved in Hepes buffer (10 mM, pH 7.0) at a final concentration of 20 m g/ml.
        2. Cationic polymers are dissolved in pure water (2.5 mg/ml).
        3. An appropriate volume of cationic polymer solution is added at room temperature to DNA by rapid addition using a Gilson. This should be performed in a polystyrene container. The solution is mixed once by gentle inversion or shaking. IT SHOULD NOT BE VORTEXED. The solution is then left at room temperature for at least 30 mins. There should be no sign of precipitation. If precipitation is seen the procedure must be repeated, with less aggressive mixing. (Simple polyelectrolyte complexes are now ready for use).
        4. For polymer coating, reactive polymer (e.g. pHPMA-ONp) is dissolved in pure water (pH >6.5, 5 mg/ml) and added to complexes at a final concentration of 200 m g/ml (i.e. 40 m l/ml).
        5. The reaction is initiated by addition of 10% volume of Hepes buffer (pH 7.8, 0.5M), and mixed by gentle shaking (no vortexing) frequently. The mixture is then placed on ice. Reaction can be monitored by measuring disappearance of intact ester at 274 nm.
        6. Targeting agent (eg. transferrin) is added in water at desired concentration (eg. 10 � 1000 m g/ml) after 1 h (for ONp esters; for NHS esters we are still determining optimum time). The solution is gently mixed and placed at 4?C overnight.
        7. After 12-16h the reaction is finished by addition of aminoethanol (stock solution is prepared by diluting 2 m l aminoethanol to 1 ml in water, and then adding 2 m l to each ml of complexes; this gives a final aminoethanol concentration of 0.0004% vol/vol).
        8. Excess hydrophilic polymer or conjugates not containing DNA can be removed by dialysis using a high molecular weight cut-off membrane or by membrane filtration (eg. Ultrafree MC filters, 300 kDa cut off, Sigma Chemical Co., Poole, U.K)

         

        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