• 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

        Yeast Transformation (high efficiency)

        互联网

        755

         

        Yeast Transformation (high efficiency)

        Linda Hoskins/Hahn Lab

        Last modified Wed, Jul 5, 2000

        This more involved method gives higher transformation efficiency than the semi rapid method. Use DMSO if transforming a library to yeast and/or transforming a mutant strain which transforms poorly using the LiOAc method.

        1. Inoculate 5 mls YPD with a single yeast colony. Grow O/N at 30 degrees C.

        2. Add 0.5 mls culture to 4.5 mls fresh media, check A660. Add suitable amount of cells to 60 mls fresh media to give A660 = 0.2 (2 x 106 cells/ml). Grow to A660 = 1.0 (2 x 107 cells/ml), takes approximately 5 hours.

        3. Spin down 50 mls cells, wash in 10 mls sterile water, re centrifuge, resuspend in 1 ml sterile water. Transfer to 1.5 ml sterile microfuge tube, spin down, resuspend in 1 ml sterile TE / LiOAc (made fresh from 10X TE [0.1M Tris-HCl, 0.01M EDTA, pH 7.5] and 10X LiOAc [1M LiOAc pH 7.5, adjusted with diluted acetic acid]). Spin down, resuspend in 0.25 mls TE / LiOAc (4 x 109 cells/ml).

        4. Mix 50 ul yeast cells with transforming DNA and 5 ul single stranded carrier DNA (10 mg/ml, boiled and quick chilled on ice) in a 1.5 ml microfuge tube.

        5. Add 300 ul sterile PEG (40% PEG 4000, 1X TE, 1X LiOAc, made fresh from sterile 50% PEG 4000, 10X TE, and 10X LiOAc). Mix thoroughly.

        6. Incubate at 30 degrees C for 60 minutes with occasional gentle shaking.

        7. Add 40 ul DMSO, mix thoroughly. (This increases transformation 5-10 fold.)

        8. Heat shock at 42 degrees C for 15 minutes.
         

        9. Microfuge 10 seconds, remove supernatant. Resuspend in 1 ml 1X TE. Microfuge 10 sec. Resuspend in 1 ml 1X TE. Plate 200 ul on selective media.

         

        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