摘要
通过研究影响转化效率的诸因素,最终找到一种快速、高效酵母完整细胞转化法。整个转化步骤能在1.5h内完成。在实验中发现:小牛胸腺DNA的加入,在加入DNA后随即加入PEG4000;将42℃热冲击时间从5min延长到25min;热冲击后直接将转化混合物涂布到选择性平板上能得到高效转化效率。所用四种类型质粒的转化效率如下:YRp:3.5—7.2×10~4(线性pCN60/BamHI:1.6×10~6);YEp:1.7—2.6×10~4(线性YEp13/BamHI:8×10~4);YCp:3.7×10~4;YIp5/Stu I:7.6×10~3。用快速法检测的7株受体菌的转化能力都达到10~4个转化子/μg DNA。
A rapid and efficient yeast transformation procedure has been developed through investigation on factors affecting transformation efficiency. The manipulation of whole procedure can be done within one and half hour. High yield of transformants is obtained by adding calf thymus DNA as carrier DNA; adding PEG4000 and DNA to cell suspension simultaneously; prolonging heat shock from 5′ to 25′ and spreading the transformation mixture directly onto agar platcs after heat shock. The pretreatment of yeast intact cells with LiAc can be omilted. The transformation rates of four types of plasmid DNA were as follows, pCN60: 3.5—7.2×10~4(for linear pCN60/BamH Ⅰ: 1.6×10~5); YEp13: 1.7—2.6×10~4 (for linear YEp13/BamH Ⅰ: 8.0×10~4); RC4: 3.7×10~4; YⅠp5/Stul: 7.6×10~3. 7 recipient strains transformed by using this procedure all reached the yields of 10 transformants per microgramme DNA.
出处
《生物工程学报》
CAS
CSCD
北大核心
1990年第2期102-107,共6页
Chinese Journal of Biotechnology
关键词
酵母
细胞转化
转化效率
Tiansformation of S.cerevisiae intact cells
calf thymus DNA
heat shock
recipient strain
plasmid DNA
