Preparation of Culture of Escherichia coli for Plasmid Minipreparation

  • A small-scale plasmid isolation procedure, the miniprep, yields a sufficient amount of plasmid for the screening of clones by restriction digestion or PCR and DNA sequencing. Once a clone is confirmed for the presence of insert with the right sequence, a large amount of plasmid can be prepared by midiprep or maxiprep.
  • Miniprep requires a small amount of culture of the plasmid-containing bacterial cells that can yield sufficient bacterial cells that can be processed for plasmid isolation by miniprep protocol. 
  • Most often a single colony from the LB-agar plate is inoculated in a liquid medium. Culture is grown at 37°C in a shaker incubator overnight (12- 16 h). Grown culture corresponds to the late log phase/early stationary phase of bacterial growth and is characterized by the low content of RNA. At this stage, the grown culture has a density of 3 – 4 × 109 cells/ml and OD600  ∼ 0.8.
  • Sometimes, a well-grown colony from the LB-agar plate can directly be utilized for plasmid isolation. A well-grown colony on the LB-agar plate is prepared by restreaking a colony in a small area (0.5 – 1 cm long). This is more convenient when you need to screen a large number of colonies. It is also cost-effective and requires fewer reagents and no handling of a culture flask.
  • An antibiotic, the selection pressure, should be present at all stages of culture growth. The choice of antibiotic depends on the antibiotic-resistant gene carried by the plasmid. In absence of the antibiotic, dividing cells can lose the plasmid, resulting in low plasmid yield.

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