Plasmids DNA Extraction Guide

Plasmids are the most commonly used carrier tools in genetic engineering, which facilitates the insertion and integration of target DNA fragments to obtain recombinant plasmids, which are then introduced into host cells, and finally, the functional elements on the plasmids are used to achieve replication, transcription and expression of corresponding proteins of target DNA fragments.

Besides its widespread application in scientific research, recombinant plasmids have been widely used in the R&D and production of many types of biomedical products, such as recombinant protein drugs, plasmid DNA vaccines, gene therapy drugs, and CAR-T drugs. At present, plasmids are a basic tool in implementing genetic engineering.

The acquisition of high-quality plasmids is a guarantee for the success of downstream experiments. Plasmid extraction mainly includes three basic steps: the propagation of plasmid DNA in bacterial cells, the lysis release of plasmid DNA from bacterial cells, and the separation and purification of free plasmid DNA in the lysate. The alkaline lysis method is the most common plasmid DNA extraction method. It is separated according to the difference in denaturation-renaturation rate caused by the topological structure difference between covalently closed circular plasmid DNA and linear genomic DNA. It has the following features: high yield, wide application, and rapid extraction.

After plasmid extraction, the quality of the extraction can be assessed using a variety of methods. The general indicators for evaluating the effect of plasmid extraction include yield, purity, the proportion of supercoiled plasmids and endotoxin content. Ultraviolet spectrophotometry is generally used to determine the yield and purity of plasmids. When the measured value is OD260/280=1.7-1.9 and OD260/230>2, it means that the plasmid purity is good; The band type, size and brightness in the gel can be used to judge the concentration, proportion, and purity of supercoiled plasmids (mainly genomic DNA, RNA, and protein residues). Ideally, plasmid samples will show a single supercoiled band in agarose gels, but in most cases, 2 or 3 bands will appear. This is caused by the different migration rates of plasmids in gel electrophoresis. The supercoiled plasmid runs the fastest, and the brighter the band, the better the integrity of the plasmid. In applications that require high purity of plasmid DNA, endotoxin level is also a very important indicator. The determination of endotoxin content is usually quantitatively detected by the gold standard Limulus reagent method. When the endotoxin content is less than 0.1EU / µg, it is usually regarded as an endotoxin-free plasmid.

So, how to extract high-quality plasmids and improve the success rate of downstream experiments? Experience shows that plasmid quality is affected by many factors, in addition to the performance of the kit itself, it is also related to the host strain, medium type, culture conditions, plasmid type, plasmid size and copy number.

Strain Types

Most of the host strains used to extract plasmids are Escherichia coli, and the type of strain often has a greater impact on the quality of plasmid DNA extraction. DH5α and XL1-Blue are the more commonly used host strains, with high transformation efficiency and large plasmid yield, which can usually meet the preparation of high-quality plasmid DNA. Other strains, such as HB101, will release a large number of carbohydrate metabolites during lysis. If there are too many residues, the subsequent enzymatic reactions will be inhibited, which will adversely affect the quality of the extracted plasmid DNA. In addition, host strains with higher endonuclease activity will reduce the yield of plasmid DNA. Therefore, the type of host strain needs to be selected according to the specific use.

Plasmid Types

Both the size and copy number of the plasmid affect the plasmid yield to a certain extent. In general, the larger the plasmid or the larger the inserted DNA fragment, the lower the yield of plasmid DNA. The copy number of plasmids in bacteria is mainly determined by the replication properties of the plasmids themselves. For low-copy plasmids, more bacterial volume is often required to obtain higher plasmid concentrations. Moreover, in some bacterial strains, chloramphenicol can be added during the culture stage to improve the plasmid yield of host cells.

The Input of Bacterial Liquid

The final cell concentration of bacterial culture varies widely due to the influence of the trophic type of bacterial growth, growth conditions (oscillation speed, temperature, time), host strain or plasmid insertion type, etc. According to empirical value, 1 litre of E. coli culture solution with OD600 of 1 contains 1×1012 cells, and the wet weight of cells is about 1.5-1.8g. Since the volume of bacterial liquid cannot reflect the concentration and weight of bacterial cells, and the determination of cell weight has certain limitations, the product of two easily measurable variable values is used as the standard for determining bacterial volume before extracting plasmids (ODV = absorbance value of bacterial liquid). OD600 × volume of bacterial solution V). Taking the Genfine Plasmid Extraction Kit as an example, the correlation between the ODV value of the bacterial sample and the plasmid yield was tested, and the results showed that the plasmid DNA yield was significantly correlated with the number of input bacteria. Too much bacterial input can easily lead to insufficient bacterial lysis, and the yield of plasmid decreases. Therefore, selecting an appropriate amount of bacterial solution is a key factor in improving the yield of plasmids.

Bacterial Culture Conditions

LB (Luria-Bertani) medium is recommended for the culture of conventional high-copy plasmid host bacteria. For optimal conditions for bacterial growth, use an Erlenmeyer flask with at least 3 times the volume of medium to provide sufficient oxygen saturation. 2 x YT, TB (Terrific Broth) and other nutrient-rich media can also be selected for the cultivation of high-copy plasmid host bacteria. In addition, antibiotics must be added for selection culture when the host strain is inoculated into the medium. The shaking speed, temperature and time of the shaker all have a significant impact on bacterial growth. The shaking culture time of the bacteria should not be too long, as excessive growth will cause the death and lysis of the bacteria cells, resulting in the loss or mutation of the plasmid DNA. In order to extract high-quality plasmids, it is necessary to find the optimal conditions for bacterial amplification and reproduction.

Endotoxin Removal

Endotoxin is a general term for a class of lipopolysaccharide substances present on the cell wall of Gram-negative bacteria, and the unit is expressed in EU. Endotoxins are released in small amounts during bacterial cell growth, and large amounts are released when cells die or lyse in large numbers. Free endotoxin molecules activate the mammalian immune system to produce an inflammatory response. Therefore, for endotoxin-sensitive cell transfection experiments or animal in vivo experiments, it is necessary to remove endotoxin as much as possible when preparing plasmids to ensure high cell transfection efficiency and eliminate the interference of endotoxin in vivo experiments. According to the different experimental needs, choosing the corresponding plasmid extraction kit can get twice the result with half the effort. Genfine Plasmid Extraction Kit introduces optional endotoxin removal steps to meet different plasmid application scenarios. For endotoxin-insensitive cells, the endotoxin removal step can be omitted directly; for endotoxin-sensitive cells, the endotoxin removal step can be optionally added, which is simple and convenient for dual use in one box.

Extraction and Purification Operations

There are many methods to lyse bacterial cells and release plasmids, such as boiling method, gradient centrifugation, alkaline lysis method, etc. Due to its simple operation and mild treatment method, the alkaline cracking method is widely used in the market. To obtain high-quality plasmid DNA, the cell lysis step must not be ignored. Do not shake vigorously after adding SDS-NaOH, which will easily cause the plasmid DNA to break, affecting the integrity, and easily mixed with small fragments of genomic DNA, affecting the purity of the plasmid. A variety of methods have been developed for purifying plasmids, such as the salting-out method, silica gel media adsorption method, anion exchange resin method, etc. The salting-out method generally involves toxic reagents, and the operation is not friendly. The yield and purity of plasmid obtained by the anion exchange resin method are good, but the cost is higher. The most widely used is the silica gel media adsorption method. The plasmid yield and purity prepared by this method are better, and the cost is relatively low, which can meet the needs of most customers. When purifying the plasmid, the adsorption capacity of the adsorption column in different products to the plasmid DNA needs to be considered. After washing with the rinsing solution, centrifuge to remove residual liquid as much as possible to ensure that no ethanol remains in the plasmid. Genfine Plasmid Extraction Kit Plasmid Extraction Kit has excellent cell lysis performance and plasmid purification performance.

Preservation Method

In addition to the quality of the extracted plasmids, care should also be taken to provide a suitable storage environment to prevent plasmid degradation from affecting downstream applications. Generally, plasmids can be stored in TE solution for short-term storage at 4°C, and at -80°C or -20°C. Long-term preservation. In addition to storing the plasmid directly, the host bacteria containing the plasmid should also be stored in glycerol, which can be stored for a long time at -80°C or in liquid nitrogen.

Of course, in addition to the above-mentioned operating points, a reagent with convenient operation and high extraction efficiency are also essential. Genfine Biotech is a national high-tech biotechnology enterprise integrating R&D, production, sales, and technical services. It is committed to providing stable, reliable, and cost-effective product solutions and professional technical services for scientific research, industrial medical and biomedical fields. Genfine Plasmid Extraction Kit has been carefully designed and optimized, which can greatly improve the quality of plasmid extraction and solve the problems of low yield, low purity and high endotoxin that may be encountered in customer experiments. It is a well-deserved experimental helper.

Product information: FinePure EndoFree Plasmid Mini Kit

High yield and purity: unique glass cellulose membrane adsorption technology, efficient and specific binding of plasmid DNA.

  • Flexible endotoxin removal steps: It contains endotoxin removal buffer, which can be selectively added or removed according to different downstream needs. The operation is simple, convenient, and versatile.
  • Timesaving: the whole extraction process is about 50 min.
  • Versatile: This kit can meet the requirements of downstream cloning, enzyme digestion, sequencing, and cell transfection experiments.

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