Key steps in the plasmid purification protocol

Now that plasmid purification is no longer a problem, do you understand the principle of plasmid purification after doing this plasmid extraction? Do you know which steps are critical to the success of plasmid purification?

Simple principle of QIAGEN plasmid extraction:

After lysing the bacteria under alkaline conditions, the lysate was added to the QIAGEN-tip at the indicated salt concentration. Plasmid DNA will selectively bind from RNA, proteins and other cellular contaminants.

1. Preparation of cell lysate

Cell yield is largely dependent on the quality of cell lysis. Therefore, the preparation of clarified cell lysates is an important part of QIAGEN's purification protocol, and QIAGEN has carefully designed the relevant cleavage conditions.

After the culture was harvested and resuspended, the bacterial cells were lysed using NaOH-SDS (P2 buffer) in the presence of RNase A. SDS dissolves phospholipids and protein components in the cell membrane, causing the cells to cleave and release the contents. NaOH denatures chromatin and plasmid DNA as well as proteins. Optimized lysis time allows for the release of the most plasmid DNA in the cell, but does not release chromosomal DNA that binds to the cell wall, thus minimizing the time it takes for the plasmid to be exposed to denaturing conditions. Excessive time of alkaline treatment may result in irreversible denaturation of plasmid DNA formation. Plasmids in this state move faster in agarose gels and are resistant to degradation by restriction enzymes. The lysate was then neutralized by the addition of acidic potassium acetate (P3 buffer). High salinity causes precipitation of KDS*, and denatured proteins, chromosomal DNA, and cell debris are subsequently captured by the salt-detergent complex. Plasmid DNA, due to its smaller size and covalent closure, is re-reprogrammed correctly and remains in the liquid phase. Since any residual SDS in the lysate will inhibit the binding of DNA to QIAGEN resin, the lysate must be mixed slowly and thoroughly to ensure complete precipitation of the detergent.

* Potassium lauryl sulfate.

The principle of isolating plasmids from chromosomal DNA is based primarily on the co-precipitation effect between the chromosomal DNA bound by the cell wall and the insoluble complex of salts, detergents and proteins. The plasmid DNA remains in the clear liquid phase above. Intense treatment during the lysis process will sever the bacterial chromosomes, causing contamination of the chromosomal DNA fragments in the supernatant. Subsequent reaction conditions will not chemically distinguish between plasmid DNA and chromosomal fragments, so these two components will not be separated by QIAGEN resin and will be eluted together at the same salt concentration. RNase A added at the beginning of the experiment was able to effectively degrade the RNA components released during alkaline lysis. The resulting RNA fragments do not bind to QIAGEN resin at the salt concentration and pH conditions at which the lysate is placed. The pellets are removed by centrifugation or by the use of QIAfilter Cartridge, and the resulting clear lysate can be loaded directly into the QIAGEN-tip. The clarity of the lysate in this stage is important to ensure good fluidity of the liquid phase and ultimately to ensure complete removal of the DNA product from the protein.

2. Clarify the bacterial lysate using QIAfilter Cartridge

QIAfilter Cartridge is a special type of filtration device designed to replace the centrifugation step of bacterial alkaline lysate. After the culture was precipitated, the bacterial cells were lysed in NaOH-SDS and added with acidic potassium acetate for neutralization, followed by direct incubation into the QIAfilter Cartridge. The lysate can pass through the filter in just a few seconds, with a clear liquid phase. Insoluble complexes comprising chromosomal DNA, salts, detergents and proteins formed in the neutralization reaction are completely eliminated during this process. The purification of bacterial lysates by QIAfilter Cartridge is more effective than conventional centrifugation. In addition, small SDS precipitates that cannot be purified by centrifugation can also be completely removed by QIAfilter Cartridge.

3. DNA binding and washing in QIAGEN-tip

The clear lysate was loaded into a pre-equilibrated QIAGEN-tip and the binding reaction was completed by gravity flow. The salt and pH conditions of the lysate, together with the excellent selectivity of the QIAGEN resin, ensure the specificity of plasmid DNA binding, and the degraded RNA, cellular proteins and metabolites do not bind and flow out with the liquid phase. The QIAGEN-tip is further washed with a medium salt buffer (QC buffer) to remove any form of contaminant residues such as trace amounts of RNA and proteins (e.g., RNase A) without any effect on the binding of the plasmid DNA. QC buffer also interrupts non-specific interactions and removes nucleic acid binding proteins without the introduction of phenol. Low concentrations of alcohol in the wash buffer eliminate non-specific hydrophobic interactions, thereby increasing the purity of the bound DNA. Since then, plasmid DNA has been efficiently eluted from the QIAGEN-tip in high salt buffer (QF or QN buffer).

4. Desalination and concentration by centrifugation

The eluted plasmid DNA was desalted and concentrated by isopropanol precipitation. The precipitation reaction is carried out at room temperature to minimize the coprecipitation effect of the salt. After centrifugation, the DNA pellet was washed with 70% ethanol to remove residual salts; while isopropanol was replaced with more volatile ethanol to facilitate subsequent removal of the liquid phase. The purified DNA can be used for transfection, sequencing, labeling, cloning, and any other experimental procedure by a simple air drying step and re-dissolution of a small volume of pH 8.0 TE buffer or pH 8.5 Tris•Cl. .

5. Desalination and concentration using the QIAprecipitator Module

The eluted plasmid DNA was mixed with isopropyl alcohol and added to the QIAprecipitator Module in the kit using a syringe. The module is capable of capturing DNA precipitates in a mixture of isopropanol liquids. It is recommended to use ethanol for additional washing steps to maximize DNA purity. The DNA precipitate obtained by the QIAprecipitator forms a thin layer structure, and by simply pushing it with a syringe, air can be passed through the QIAprecipitator to achieve sufficient drying of the DNA while removing alcohol. The DNA is then eluted from the QIAprecipitator into a microcentrifuge tube using the TE buffer supplied by the kit. Any commonly used buffer or water can be used as an alternative to elution. If eluted with pure water, the DNA should be stored at a temperature of –20 ° C because the DNA will degrade in the absence of a buffer system and a chelating agent. The DNA product thus obtained is suitable for transfection, sequencing, labeling, cloning, and any other subsequent application.

The above information is from QIAGEN:

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