• 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

        Raft culture media (aka Green’s media)

        互联网

        1438

         

        Raft culture media (aka Green’s media)

        3 parts DMEM (including glutamine or glutamax)
        1 part Ham’s F12
        5% FCS
        various supplements (not everybody uses the same supplements, some don’t use any!)

        The following are the supplements I have used. I doubt the necessity of the last three but have not got round to trying without them.

        Adenine (Sigma A8626), MW=135, final concentration=180m M,  working stock=45mM, Dilution factor=250.

        Dissolve in 0.05N HCl to 45mM.
        Store working aliquots at �20C.

        Insulin (Sigma I6634), MW=5733, final concentration=5m g/ml,  working stock=5mg/ml, Dilution factor=1000.

        Dissolve in acidified H2 O (0.1ml glacial AcCOOH + 9.9ml H2 O)
        I had to add more glacial acetic acid to make it dissolve.
        Store working aliquots at 4C.

        Hydrocortisone (Sigma H0135), MW=362, final concentration=400ng/ml,  working stock=1mg/ml, Dilution factor=2500.

        Follow instructions with vial (dissolve in EtOH then add equal volume of H2 O)
        Store working aliquots at -20C.

        Transferrin (Sigma T2036), final concentration=5m g/ml,  working stock=500m g/ml, Dilution factor=100.

        Dissolve in media (DMEM or KGM)
        Store working aliquots at -20C.

        Pen and Strep

        Fungizone

        T3 (aka 3,3’,5-triiodo-L -thyronine) (Sigma T6397), MW=673, final concentration=0.2nM,  working stock=1m M, Dilution factor=7500.

        Dissolve in 1N NaOH to 1mg/ml.
        Dilute 1000-fold in 0.1N NaOH.
        Store working aliquots at -20C.

        Cholera toxin (Sigma), MW=87kD, final concentration=0.1nM,  working stock=50m g/ml, Dilution factor=5750.

        Careful!

        EGF (Sigma E1264), final concentration=5ng/ml,  working stock=100m g/ml, Dilution factor=20000.

        Dissolve in media (DMEM or KGM)
        Store working aliquots at -20C.

        (also, I have seen in some papers that people add ascorbic acid immediately before use. I have never tried this.)

         

        Example (for a little over 100ml raft medium)

        70ml DMEM
        25ml F12
        5ml FCS

        Add the following to the FCS and sterile filter before adding to the media;

        400m l of Adenine (45mM)
        100m l of Insulin (5mg/ml)
        40m l of Hydrocortisone (1mg/ml)
        1ml of Transferrin (500m g/ml)
        13.5m l of T3 (1m g/ml)
        18m l of cholera toxin (50 m g/ml)
        5m l of EGF (100 m g/ml)

        also;

        200m l fungizone (250 m g/ml)
        1ml Pen and Strep (100x strength (10000 units/ml and 5000 m g/ml))

         

        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