- Amplification of plasmid is desirable for many applications including gene cloning, DNA sequencing, transfection, and probe preparation. Fastest and routinely used method to amplify plasmid is to introduce plasmid in an appropriate strain of E. coli e.g. DH5α (the process is called transformation), grow them to a suitable culture volume, and finally, extract plasmid from them (the process called plasmid isolation).
- Alternatively, E. coli DH5α harboring a plasmid can also be revived from the stored stocks e.g., glycerol stock or stab culture, if available. Cells obtained from stored stock can be either streaked or plated on an antibiotic containing solid LB-agar plate.
- Plasmid copy number and culture volume are the two most important parameter which predicts the quantity of the plasmid extracted at the end of the isolation process. Comparatively, large culture volume is required for low copy number plasmid.
- Plasmid copy number can be increased by chloramphenicol treatment. Several rich growth media can also be used to grow bacteria. These media support high cell density due to nutrient enrichment.
- Depending on the initial culture volume, plasmid isolation methods are called miniprep (1-5 ml culture volume), midiprep (25-50 ml culture volume), and maxiprep (100-500 ml culture volume).
- Generally miniprep yields sufficient amount of plasmid for applications like the screening of clones for the presence of insert, DNA sequencing etc. Other applications, like probe preparations, plasmid distribution, transfection, etc., can require a large quantity of plasmid (midiprep or maxiprep).
- For miniprep, a single colony from the LB-agar plate is inoculated into a antibiotic-containing liquid medium. Culture is grown at 37°C in a shaker incubator overnight (12- 16 h). Grown culture corresponds to late log phase/early stationary phase of bacterial growth and is characterized by low content of RNA. Incubating culture for a long time can cause the death of bacteria, which can result in low yield of plasmid. Sometimes, a well-grown colony from the LB-agar plate can directly be utilized for plasmid miniprep.
- For a large amount of culture which is required for midiprep and maxiprep, initially, a starter culture is prepared by inoculating a small amount of culture medium (2 – 10 ml) with a single colony. When the culture reaches mid- to late-exponential growth phase (takes 8 – 12 h), culture is diluted in a ratio of 1:100 to 1:1000 to prepare large culture volume for midiprep and maxiprep.
- All plasmid vectors carry at least one antibiotic resistance gene, which enables bacteria to survive and grow in presence of a respective antibiotic. Antibiotic functions as a selective marker which allows growth of only plasmid containing E. coli cells. In absence of antibiotic, bacteria will lose the plasmid, which will result in low or no yield.