Suga Minoru, Hatakeyama Toyomasa
Department of Materials and Biosystem Engineering, Toyama University, 3190 Gofuku Toyama-City, Toyama 930-8555, Japan.
Yeast. 2005 Jul 30;22(10):799-804. doi: 10.1002/yea.1247.
A rapid, simple, convenient, and highly efficient transformation of the fission yeast Schizosaccharomyces pombe has been developed. Freezing fission yeast cells in glycerol, a permeating cryoprotectant, with lithium acetate improved remarkably the transformation efficiency by one to two orders of magnitude. The optimum concentration of glycerol was found to be 30%, which is higher than that (10-15%) in the conventional cryopreservation of yeast cells. Glycerol not only played a role in cryopreserving the competent cells but also improved the transformation efficiency of the process. The thawed cell suspension with glycerol and lithium acetate was immediately mixed with carrier DNA, plasmid DNA and polyethylene glycol. Next, the mixture was heat shocked and directly spread on a selection plate. This simple procedure yielded more than 10(6) transformants/microg plasmid DNA, reducing the time required to only 20 min in total, including the thawing time. Furthermore, the frozen competent cells were stored long-term for more than 3 months without any significant loss of efficiency.
已开发出一种快速、简单、便捷且高效的粟酒裂殖酵母转化方法。用渗透型冷冻保护剂甘油和醋酸锂冷冻裂殖酵母细胞，可使转化效率显著提高一到两个数量级。发现甘油的最佳浓度为30%，这高于酵母细胞传统冷冻保存中的浓度（10 - 15%）。甘油不仅在冷冻保存感受态细胞中起作用，还提高了该过程的转化效率。将含有甘油和醋酸锂的解冻细胞悬液立即与载体DNA、质粒DNA和聚乙二醇混合。接下来，混合物进行热激处理并直接涂布在选择平板上。这个简单的步骤每微克质粒DNA可产生超过10⁶个转化子，总共所需时间减少到仅20分钟，包括解冻时间。此外，冷冻的感受态细胞可长期保存超过3个月，而效率没有任何显著损失。
