PEG Purification Techniques of Plasmid DNA

Plasmid DNA extraction is a laboratory technique used to isolate and purify plasmid DNA from bacterial cells. Plasmids are small, circular pieces of DNA that are separate from the chromosomal DNA of bacteria. They often contain genes that confer advantageous traits, such as antibiotic resistance or the ability to produce a desired protein. Plasmid DNA extraction involves breaking open bacterial cells to release the plasmids, separating the plasmids from other cellular components, and purifying the plasmid DNA using various techniques such as centrifugation, column chromatography, and precipitation. The purified plasmid DNA can then be used for various applications, such as cloning, gene expression studies, and genetic engineering.

Common Plasmid DNA Purification Techniques

Large quantities of extracted plasmid DNA generally require further purification. Commonly used plasmid DNA purification techniques are column chromatography and cesium chloride gradient centrifugation. Column chromatography involves passing the plasma DNA through a resin or gel column that separates DNA based on its size, charge, and other physical properties. This method can be very effective in removing impurities and obtaining highly pure plasma DNA. Cesium chloride gradient centrifugation involves layering a cesium chloride solution with the plasma DNA sample and centrifuging it at high speeds. The cesium chloride forms a density gradient, which separates the DNA based on its density. This method can also be very effective in obtaining highly pure plasma DNA.

Fig. 1. Plasmid DNA was purified by PEG precipitation (J. Chromatogr. A. 2010, 1217: 1429-1436).

Purification of Plasmid DNA by PEG Precipitation

Of these, polyethylene glycol (PEG) precipitation is the best method for initial plasmid DNA purification. PEG is a widely used non-ionic polymer, which is an important class of precipitants developed in the 1960s. Based on the precipitation of polymers (depending on the concentration of the polymer and the molecular weight of the precipitated material), Laurent et al. (1967) proposed that the precipitation mechanism of PEG is mainly through steric repulsion to force particles in the liquid (including biomacromolecules, viruses and bacteria) to gather together for precipitation reactions. PEG was first used to purify immunoglobulin (IgG) and to precipitate some bacterial viruses. Later, it was gradually applied to the separation and purification of nucleic acids and enzymes, especially the separation of plasmid DNA with PEG.

General steps for PEG purification of plasmid DNA:

1. Add 8.0 mL of 4 M NaCl and 40 mL of 13% PEG 8000 (w/v) to 50 mL of miniprep plasmid and incubate on ice for 20-30 minutes.

2. Centrifuge at maximum speed for 10 minutes at 4°C, then gently aspirate the supernatant to collect the pellet.

3. Use 500 liters of 70% ethanol to rinse the pellet. At this point, the pellet's color turns milky white.

4. Gently drain the supernatant from the pellet and give it another 500 mL of 70% ethanol rinse.

5. Drain the ethanol and leave the tube inverted until the pellet has lost all traces of the ethanol.

6. Mix 20 to 30 mL of sterile water with the pellet to dissolve it.

7. Before submitting the plasmid for sequencing, perform an agarose gel analysis to determine its integrity and purity.

8. A high-purity, high-quality plasmid is now available.

The advantage of this operation is that the operating conditions are mild and it is not easy to cause denaturation of biological macromolecules. At the same time, this technology has extremely high precipitation efficiency, and a small amount of PEG can precipitate a considerable lot of biological macromolecules.

In addition to being widely used in plasmid DNA purification, PEG can also be used in protein purification technology and radiolabeling technology. As a leading supplier of PEG, BOC Sciences can provide high-purity and low-dispersion PEG products for your nucleic acid purification and protein purification. Our PEG products have been widely used in various applications, including chromatography, electrophoresis, and cell culture. We offer a range of PEG products with different molecular weights and functional groups to meet your specific needs.

Reference

1. Barbosa, H.S.C. et al. Dual affinity method for plasmid DNA purification in aqueous two-phase systems. J. Chromatogr. A. 2010, 1217: 1429-1436.