• 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

        Frozen Yeast TRAFO Protocol

        互联网

        1137

         

        This Protocol allows You to prepare Frozen Yeast cells that are competent for transformation after thawing

        After Dohmen et al. (1991) Yeast 7: 691-692. See Schiestl et al (1993)

         


         

        The ability to make yeast cells competent for transformation in advance allows those laboratories working with few strains to prepare large batches of cells for later transformation. This is convenient for laboratories using the two-hybrid and other similar systems. Yeast cells can be made competent for transformation by treatment with ethylene glycol and dimethyl sulfoxide (DMSO) and then frozen in small aliquots and stored at - 70o C. While the highest transformation efficiencies are obtained with freshly grown cultures, the moderately efficient transformation of frozen competent cells saves time. This procedure has been reported to produce up to 105 transformants / µg plasmid DNA (Dohmen et al. 1991).

         



         

        1. Grow cells in YPAD (10 ml per transformation) to an OD600 or 0.6 to 1.0. This represents a cell density of approximately 0.6 - 1 x 107 cell/ml.
        2. Wash the cells in 0.5 vol of 1.0 M. sorbitol, 10 mM Bicine-NaOH (pH 8.35), 3% ethylene glycol, 5% DMSO (Solution 1), and resuspend in 0.02 vol of the same solution.
        3. Freeze the 0.2 ml aliquots slowly using a Nalgene Cryo 1o C freezing container (Cat. No. 5100-0001) and store at -70o C until needed.

          TIP:

          Slow freezing results in good viability.

           

        4. Add 0.1 - 5 µg of plasmid DNA and 50 µg of single stranded carrier DNA (10 mg/ml) in a maximum volume of 20 µl on top of the frozen cell suspension.
        5. Place in a 37o C water bath and mix every 10 - 15 sec until the solution begins to melt. Remove from water bath and mix until melting is complete.
        6. Add 1.4 ml of Solution 2 (40% PEG1000 (Roth, Karlsuhe, Germany), 0.2 M Bicine-NaOH (pH 8.35)) and mix by vortexing for 1 min.
        7. Incubate at 30o C for 1 hr.
        8. Spin down the cells at 3000 x g for 5 sec and resuspend the cell pellet in 1.0 ml of Solution 3 (0.15 M NaCl, 10 mM Bicine-NaOH (pH 8.35)).
        9. Plate an appropriate amount onto SC ommission medium into a puddle of Solution 3.

        Preparation of frozen competent cells from the two-hybrid strain PJ69-4A using the method above routinely gave results between 0.9 - 1.0 x 104 transformants/µg.

         

        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