Gietz R D, Schiestl R H, Willems A R, Woods R A
Department of Human Genetics, University of Manitoba, Winnipeg, Canada.
Yeast. 1995 Apr 15;11(4):355-60. doi: 10.1002/yea.320110408.
An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.
一种改进的醋酸锂（LiAc）/单链DNA（SS-DNA）/聚乙二醇（PEG）方法（该方法每微克质粒DNA可产生超过1×10⁶个转化子）以及Schiestl和Gietz（1989年）描述的原始方法被用于研究LiAc/SS-DNA/PEG转化机制的相关方面。当在50微克SS载体DNA存在的情况下，用100纳克质粒DNA转化1×10⁸个细胞时，观察到最高的转化效率。每次转化的转化子产量随质粒增加至5微克呈线性增加。在42℃进行20分钟的热激对于获得最大产量是必要的。发现PEG能将载体DNA和质粒DNA都沉积到细胞上。与双链（DS）载体相比，SS载体DNA与细胞结合更有效，并且能使³²P标记的质粒DNA结合更紧密。LiAc/SS-DNA/PEG转化方法不会导致细胞融合。DS载体DNA在转化反应中与DS载体DNA竞争。SS质粒DNA与SS和DS载体DNA组合时转化细胞的效果较差。LiAc/SS-DNA/PEG方法被证明比已知的其他使细胞可转化的处理方法更有效。本文讨论了LiAc/SS-DNA/PEG方法的转化机制模型。
