Institute for Advanced Biosciences, Keio University, Yamagata, Japan.
Yeast. 2012 Aug;29(8):311-22. doi: 10.1002/yea.2911. Epub 2012 Jul 5.
Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality.
传统的酵母提取方案是为相对快速生长的低细胞密度的实验室菌株培养而开发的，通常不适用于频繁取样的培养物。因此，这些方案通常对生长缓慢的培养物或非实验室菌株的效率不高。我们开发了一种结合机械和化学破坏的方法，使用剧烈的珠磨，可以始终如一地破坏酵母细胞（>95%），无论细胞周期和代谢状态如何。使用这种破坏技术与淬灭相结合，我们开发了 DNA、RNA 和蛋白质提取方案，这些方案针对大量来自生长缓慢的高密度工业酵母培养物的样品进行了优化。此外，还最小化了样品体积、昂贵试剂/酶的使用、处理时间和孵育时间。我们使用重复/时间序列提取测试了我们方法的重现性，并将其与常用方案或市售试剂盒进行了比较。此外，我们还利用简单的流式细胞术方法来估计线粒体 DNA 拷贝数。基于这些结果，我们的方法显示出更高的重现性、产量和质量。
