科研之友
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摘要
The present study was designed to achieve high DNA transformation efficiency of E. coli by using the combination of chemical and physical transformation methods. The effect of low growth temperatures, osmotic agents, reducing agents, field strength, pre- and post-electroporation heat shock treatments; surfactant and different combinations of some of these factors were studied in E. coli. Cells grown at low temperature required longer time to reach required density and no improvement in transformation efficiency observed. Osmotic agents in growth medium changed the growth trend of bacteria and their presence in transformation mixture proved helpful in achieving up to 10(8) transformants/mu g of plasmid DNA. The addition of beta-mercaptoethanol in growth and transformation mixture improved transformation efficiency by 10 folds compared to control. Higher field strength of 12.5 kV cm(-1) was found to increase the transformation efficiency. Delay in electroporation after mixing plasmid DNA and competent cells as in case of pre- electroporation heat shock and use of PEG decreased the transformation efficiency. Pre- and post-electroporation heat shock decreased cell survival as well as transformation frequency. The use of sucrose, glycine and beta-mercaptoethanol (in growth and subsequent transformation media), and field strength of 12.5 kV cm(-1) positively affected the transformation efficiency.
- 出版日期2014
