Basic Steps in Plasmid Isolation

Plasmid isolation is a common task in molecular biology laboratories. Tasks as simple as amplifying a plasmid vector to complex tasks like gene therapy and gene manipulation involves plasmid isolation steps. 

Several methods have been described to isolate plasmid from E. coli. These methods take advantage of plasmid small size and its covalently closed circular structure to separate plasmid DNA from genomic DNA. All these methods have the following common steps:

• Culturing of plasmid-containing E. coli cells
• Harvesting
• lysis
• Purification of plasmid

Step 1: Culturing of plasmid-containing E. coli cells

• A sufficient amount of plasmid-containing bacterial cells is the starting point of all plasmid isolation procedures. Usually, a liquid culture that is prepared by inoculating a single colony of E. coli is used for harvesting bacterial cells for plasmid isolation. Depending on the needs, a liquid culture can be scaled up to any volume. The term “miniprep” (2- 5 ml initial culture volume), “midiprep” (10 – 25 ml initial culture volume), and “maxiprep” (100 – 250 ml initial culture volume) are used for different initial culture volumes.
• A well-grown colony can also be directly taken from an LB-agar plate for the miniprep of high-copy number plasmids. It can provide enough plasmid DNA which is sufficient for few restriction digestion/PCR reactions, therefore is useful for screening of a large number of clones. 
• Many factors including E. coli strains, copy number of plasmid, initial culture volume, affect the quality and quantity of plasmid. Choosing the right method is key to obtaining a suitable quantity and quality of plasmid DNA that suits your need and the particular application. For example, transfection grade plasmid must be of high quality, devoid of any impurities.
• Several culture mediums are available to grow E. coli. LB liquid broth is most commonly used to grow E.coli DH5𝝰 for plasmid isolation. Chloramphenicol can be used to increase the yield low copy number plasmid. Other rich culture mediums, e.g., Terrific Broth, Super Broth, etc can also be used which supports better growth of E. coli than LB broth.
• Culture medium composition, culturing temperature and time, bacterial strain, shaking speed are important to grow a healthy culture of bacteria.

Step 2: Harvesting of bacteria from liquid culture

• Harvesting of bacterial cells from liquid culture is usually done by centrifugation.
• Centrifugation speed and time is critical for harvesting bacterial cells from liquid culture. Centrifugation at high speed for a long time can result in a tight pellet which could be difficult to resuspend that can result in inefficient action of lysis solution in subsequent steps. On the other hand, low centrifugation speed and less time can result in inefficient harvesting of bacterial cells from the culture. In both situations, plasmid yield will be lower than expected.

Step 3: Lysis of bacterial cells

• Harvested cells are subjected to lysis that results in the release of plasmid DNA in solution. Usually harvested cell pellet is resuspended in an appropriate buffer to help the lysis solution to have a similar action on each bacterial cell. Cell clumps in suspension result in inefficient lysis, leading to low yield of plasmid DNA. 
• Plasmid isolation methods utilize several strategies to induce lysis in bacterial cells in a highly controlled manner which include heat, detergents, alkaline condition, enzymes, and their combination. Often a gentle lysis method is preferred in case of a large plasmid.
• The boiling method of plasmid isolation allows controlled lysis resulting in the release of only plasmid DNA from the bacterial cells, other components including genomic DNA are trapped inside bacterial cells. In contrast, cell lysis in the alkaline lysis method results in all material including genomic and plasmid DNA release in the solution.

Step 4: Purification of plasmid

• Once the plasmid is in solution, strategies are used to selectively isolate it from other bacterial components. 
• The Alkaline lysis method utilizes plasmid small size and covalently closed circular structure that results in the rapid renaturation of plasmid DNA in the right order, which results in plasmid remains soluble and present in a liquid phase. On the other hand, the boiling method selectively allows the release of plasmid DNA from the bacterial cells. Subsequent filtration or centrifugation removes the insoluble impurities. The resulting solution which contains plasmid is either subjected to further purification or precipitated using ethanol or isopropanol. 
• Several purification strategies including phenol-chloroform extraction, ion-exchange or gel-filtration chromatography can be used to obtain high quality of plasmid.