ONE-STEP PREPARATION OF COMPETENT ESCHERICHIA-COLI - TRANSFORMATION AND STORAGE OF BACTERIAL-CELLS IN THE SAME SOLUTION
ONE-STEP PREPARATION OF COMPETENT ESCHERICHIA-COLI - TRANSFORMATION AND STORAGE OF BACTERIAL-CELLS IN THE SAME SOLUTION
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DOI：
10.1073/pnas.86.7.2172
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发表时间：
1989-04-01
影响因子：
11.1
通讯作者：
MILLER, RH
中科院分区：
文献类型：
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作者：
CHUNG, CT;NIEMELA, SL;MILLER, RH
我们已经开发了一种简单的一步法来制备具有活性的大肠杆菌，它使用转化和储存溶液[TSS：1。TSS是含有10%(重量/体积)聚乙二醇、5%(体积/体积)二甲亚砜和50 mM镁离子(在pH 6.5下)的LB肉汤。将细胞与等量的冰块混合2倍。TSS，并立即准备使用。遗传转化同样简单：加入质粒DNA，在4度的温度下孵育5-60分钟。C.不需要热脉冲，孵化时间为4度。C不是关键的，所以在转换过程中没有关键的计时步骤。转化的细菌是通过标准方法培养和选择的。因此，该程序省去了当前方法的离心法、洗涤法和长期孵化步骤。虽然在生长周期早期(OD500 0.3-0.4)的细胞产生最高的转化效率(107-108个转化子/微克DNA)，但在生长周期的其他阶段(包括稳定期)收获的细胞能够进行深入转化(105-107个转化子/微克DNA)。为了长期保存感受态细胞，细菌可以在不添加其他成分的情况下冷冻在TSS中。我们的程序为制备、转化和储存感受态细菌细胞提供了一种简单方便的方法。
We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solutin [TSS: 1 .times. TSs is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 .times. TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4.degree. C. A heat pulse is not necessary and the incubation time at 4.degree. C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells takern early in the growth cycle (OD500 0.3-0.4) yield the highest transformation efficiencies (107-108 transformants per .mu.g of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoint transformation (105-107 transformants per .mu.g of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.