- Plasmid isolation is an important step in many molecular biology techniques such as gene cloning, DNA sequencing, transfection, and probe preparation.
- The 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).
- If available, E. coli DH5α harboring a plasmid can also be revived from the stored stocks such as glycerol stock or stab culture. Cells obtained from stored stock can be either streaked or spread on an antibiotic-containing LB-agar plate.
- Plasmid copy number and culture volume are the two most important parameters which can influence the quantity of the plasmid extracted at the end of the isolation process. Comparatively, a large culture volume is required for a low copy number plasmid.
- Copy number of low copy number plasmids can be increased by chloramphenicol treatment.
- Usually, LB (lysogeny broth, also referred to as Luria-Bertani growth medium) is the most suitable growth medium for most laboratory strains of E. coli as most plasmid isolation protocols and commercially available kits are standardized for this. The recommended initial volumes of E. coli culture and buffers for lysis and other downstream processes in these protocols and kits are suitable for the cell density achieved by the overnight culture of E. coli in the LB medium. However, several highly rich growth media (e.g., Terrific Broth (TB)) are also available that can be used to culture E. coli. These culture media support a much higher saturation density thus helping to reduce the initial E. coli culture volume.
- 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 a 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 an antibiotic-containing liquid medium. Culture is grown at 37°C in a shaker incubator overnight (12- 16 h). The grown culture corresponds to the stationary phase of bacterial growth and is characterized by a low content of RNA. Incubating culture for a long time can induce the death of bacteria, which can result in genomic DNA contamination and low yield of plasmid in preparation. 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 the mid to late-exponential growth phase (takes 8 – 12 h), the culture is diluted in a ratio of 1:100 to 1:1000 to prepare a 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 the presence of a respective antibiotic. Antibiotics function as a selective marker that allows the growth of only plasmid-containing E. coli cells. In the absence of the antibiotic, bacteria will lose the plasmid, which will result in low or no yield.
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