• 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

        Expression Protocol in M9 Minimal Media via T7 Promoter

        互联网

        941

         

        Expression Protocol in M9 Minimal Media via T7 Promoter

        The following protocol has been used successfully to 15 N or 13 C/15 N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4 Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4 Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:
        • 64g Na2 HPO4 -7H2 O
        • 15g KH2 PO4
        • 2.5g NaCl
        • H2 O to final 1L volume and autoclave
        To prepare 500mL M9 minimal media;
        • 100mL of 5xM9 salts
        • 1 mL 1M MgSO4
        • 50 uL 1M CaCl2
        • 5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
        • 2.5 mL filter sterilized NH4 Cl (0.2 g/mL) or 0.5g dry
        • 10 mL 20% d-glucose or 2g dry
        • glass distilled & autoclaved H2 O to final volume of 500mL
        1. pH solution to 7.3 and filter sterilize (0.2u filter).
           
        2. Introduce media to a pre-autoclaved, wide-bottom (baffled) 2L flask and add ampicillin to a final concentration of 70-100 ug/ml.
           
        3. Grow 5mL overnight culture in same media to inoculate 500mL M9.
           
        4. Shake culture at 37o C until an OD600 of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).

         

        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