Transformation of Escherichia coli by Electroporation.
Transformation of Escherichia coli by Electroporation.
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DOI：
10.1101/pdb.prot101220
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发表时间：
2020-06-01
影响因子：
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通讯作者：
Sambrook, Joseph
中科院分区：
文献类型：
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作者：
Green, Michael R;Sambrook, Joseph
制备电活性细菌比用化学方法准备细胞转化要容易得多。细菌简单地生长到对数中期，冷却，离心，用冰冷缓冲液或水洗涤，以降低细胞悬浮液的离子强度，然后悬浮在含有10%甘油的冰冷缓冲液中。DNA可以通过将细菌暴露在短时间的高压放电中而立即进入细菌。或者，细胞悬浮液可以在电穿孔前快速冷冻并在-70°C下保存长达6个月，而不会损失转化效率。
Preparing electrocompetent bacteria is considerably easier than preparing cells for transformation by chemical methods. Bacteria are simply grown to mid-log phase, chilled, centrifuged, washed extensively with ice-cold buffer or H2O to reduce the ionic strength of the cell suspension, and then suspended in an ice-cold buffer containing 10% glycerol. DNA may be introduced immediately into the bacteria by exposing them to a short high-voltage electrical discharge. Alternatively, the cell suspension may be snap-frozen and stored at -70°C for up to 6 mo before electroporation, without loss of transforming efficiency.