• 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

        Joe mRNA prep

        互联网

        699

        Joe's mRNA prep
        (Audrey Gasch, Pat Brown [modified by S.M. Hettenbach])

        Oligo-dT cellulose prep

        1. Dump Ambion vial contents into 50 ml c/f tube
        2. Add 10 ml 1x NETS to vial, cap, rinse vial, dump 10 ml into c/f tube
        3. Spin tube 3000 rpm, 2 min at 4°C in swinging bucket with no brake, remove s/n
        4. Add 10 ml 1x NETS to tube, resuspend contents by swirling
        5. Spin tube 3000 rpm, 2 min at 4°C in swinging bucket with no brake, remove s/n
        6. Repeat steps 4 and 5 two more times for a total of 4 washes
        7. Add 10 ml 2x NETS and store at 4°C
        8. Prep cellulose the day before you need it

        mRNA prep

        1. Dilute 1 mg total RNA to 1 ml with 10mM Tris pH7.4 in c/f tube
        2. In a 2 ml BioRad mini column(#731-1550) sealed on bottom, mix 1 ml resuspended resin with 1 ml of 1 mg/ml total RNA from step 1. Cap and parafilm column, tape to rocker, and bind at RT for 1 hour on 5.5 setting
        3. Break bottom off column and place in 16 x125 mm test tube and allow contents to flow through
        4. Wash resin five times with 0.7 ml 1x NETS, slow flow is normal
        5. Transfer column to support and elute 2x with 0.7 ml 10 mM Tris pH 7.4 preheated to 70°C into separate 1.7 ml tubes. Elute with 150 Joe mRNA prep l of the 0.7 ml volume at a time keeping rest at 70°C.
        6. Add 0.7 ml chloroform to each tube to remove residue cellulose, mix well, spin 13,000 rpm for 1 min at RT, transfer upper phase to new tube
        7. Precipitate with 0.1 volume 3M NaOAc pH 7.0 and 1 vol isopropanol at -20°C for 1 hour
        8. Spin 13,000 rpm for 30 min at 4°C
        9. Remove s/n and air dry
        10. Resuspend both tubes in final volume of 20Joe mRNA prep l of DEPC ddH2 O
        11. Spec OD260 & OD280 on 1 Joe mRNA prep l mRNA. 1 OD260 is equal to 40 Joe mRNA prep g/ml of RNA
        12. Store at -80°C, avoid repeat freezing and thawing

         

        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