Mao X H, Cai J K
Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Chin J Biotechnol. 1990;6(2):103-10.
A rapid and efficient yeast transformation procedure has been developed through investigation of factors affecting transformation efficiency. The manipulation of the entire procedure can be done within one and one half hours. High yield of transformants is obtained by: adding calf thymus DNA as carrier DNA; adding PEG4000, carrier DNA and plasmid DNA to cell suspension simultaneously; prolonging heat shock at 42 degrees C from 5 min to 25 min and spreading the transformation mixture directly onto agar plates after heat shock. The pretreatment of yeast intact cells with LiAc can be omitted in this procedure. The transformation rates of four types of plasmid DNA were as follows: pCN60: 3.5-7.2 x 10(4) (for linear pCN60/BamHI: 1.6 x 10(5)); YEp13: 1.7-2.6 x 10(4) (for linear YEp13/BamHI: 8.0 x 10(4)); RC4: 3.7 x 10(4); YIp5/StuI: 7.6 x 10(3). Seven recipient strains transformed by using this procedure all reached the yields of over 10(4) transformants per microgramme of DNA.
通过对影响转化效率的因素进行研究，开发出了一种快速高效的酵母转化方法。整个操作过程可在一个半小时内完成。通过以下方法可获得高产的转化子：添加小牛胸腺DNA作为载体DNA；将PEG4000、载体DNA和质粒DNA同时加入细胞悬浮液中；将42℃热激时间从5分钟延长至25分钟，并在热激后将转化混合物直接铺在琼脂平板上。此方法可省略用LiAc对酵母完整细胞进行的预处理。四种质粒DNA的转化率如下：pCN60：3.5 - 7.2×10⁴（线性pCN60/BamHI为1.6×10⁵）；YEp13：1.7 - 2.6×10⁴（线性YEp13/BamHI为8.0×10⁴）；RC4：3.7×10⁴；YIp5/StuI：7.6×10³。使用此方法转化的7种受体菌株每微克DNA的转化子产量均达到10⁴以上。
