CTAB-mediated, single-step preparation of competent Escherichia coli, Bifidobacterium sp. and Kluyveromyces lactis cells.
CTAB-mediated, single-step preparation of competent Escherichia coli, Bifidobacterium sp. and Kluyveromyces lactis cells.
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DOI：
10.1016/j.mgene.2014.10.002
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发表时间：
2014-12
期刊：
影响因子：
0.7
通讯作者：
Siddiqui, Kaneez Fatima
中科院分区：
文献类型：
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作者：
Rajagopal, Kammara;Singh, Praveen Kumar;Kumar, Rajesh;Siddiqui, Kaneez Fatima
有效和可复制的转化方法取决于生物体的能力。我们开发了一种简单的方法来制备大肠杆菌、乳酸克卢维菌和双歧杆菌，该方法使用含有十六烷基三甲基溴化铵（CTAB）的缓冲液，并允许有效地吸收质粒DNA和连接反应产物。收集细胞，洗涤，与1-10 μg/ml CTAB混合，孵育，然后缓冲洗涤。为了能态细胞的长期储存，可以将细菌冷冻在10%的甘油中，而不添加其他成分。转换过程非常简单；将质粒DNA与细胞混合，在4℃下孵育5-60 min；不需要热脉冲，在4°C下的孵育时间也不重要。我们开发了一种简单快速的方法来制备大肠杆菌、双歧杆菌和乳杆菌。CTAB允许质粒的有效摄取，以及连接反应产物。在指数后期和固定阶段收获的细胞观察到相同的转化效率。CTAB处理过的细胞用于热休克、电穿孔等转化。
An efficient and reproducible method for transformation depends on the competency of the organism. We have developed a simple method for the preparation of competent Escherichia coli, Kluyveromyces lactis, and Bifidobacterium sp. by using a buffer containing cetyl trimethyl ammonium bromide (CTAB) and permits efficient uptake of plasmid DNA and ligation-reaction products. Cells are harvested, washed, mixed with 1–10 μg/ml CTAB, incubated, and followed by a buffer wash. For long-term storage of competent cells, bacteria may be frozen in 10% glycerol without the addition of other components. The transformation process is very simple; plasmid DNA and the cells are mixed and incubated for 5–60 min at 4 °C; no heat pulse is required, and the duration of incubation at 4 °C is not crucial. We developed a simple and rapid procedure for the preparation of competent E. coli, Bifidobacterium sp., and K. lactis. CTAB permits efficient uptake of plasmid, as well as ligation reaction products. The equal transformation efficiencies were observed with cells harvested at late exponential and stationary phases. CTAB treated cells are used for transformation by heat shock, electroporation, etc.