Key Steps In Plasmid Purification Protocols

Optimal conditions at every stage of the purificaton procedure ensure high yields

After lysis of bacteria under alkaline conditions, the lysate is applied under defined salt conditions to the QIAGEN-tip. Plasmid DNA is selectively bound and purified from RNA, proteins, and other cellular contaminants.

DNA yield depends on the quality of the cell lysate used. Preparation of a cleared cell lysate is therefore a critical step in the QIAGEN purification procedure, which has been carefully designed to provide optimal lysis conditions.

After harvesting and resuspension, the bacterial cells are lysed in NaOH-SDS (Buffer P2) in the presence of RNase A. SDS solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and release of the cell contents. NaOH denatures the chromosomal and plasmid DNAs, as well as proteins. The optimized lysis time allows maximum release of plasmid DNA from the cell without release of cell wall-bound chromosomal DNA, while minimizing the exposure of the plasmid to denaturing conditions. Long exposure to alkaline conditions may cause the plasmid to become irreversibly denatured. This denatured form of the plasmid runs faster on agarose gels and is resistant to restriction enzyme digestion. The lysate is neutralized by the addition of acidic potassium acetate (Buffer P3). The high salt concentration causes KDS* to precipitate, and the denatured proteins, chromosomal DNA, and cellular debris become trapped in salt–detergent complexes. Plasmid DNA, being smaller and covalently closed, renatures correctly and remains in solution. Since any SDS remaining in the lysate will inhibit binding of DNA to QIAGEN resin, the solution must be thoroughly but gently mixed to ensure complete precipitation of the detergent.

* Potassium dodecyl sulfate.

Separation of plasmid from chromosomal DNA is based on coprecipitation of the cell wall-bound chromosomal DNA with the insoluble complexes containing salt, detergent, and protein. Plasmid DNA remains in the clear supernatant. Vigorous treatment during the lysis procedure will shear the bacterial chromosome, leaving free chromosomal DNA fragments in the supernatant. Since chromosomal fragments are chemically indistinguishable from plasmid DNA under the conditions used, the two species will not be separated on QIAGEN resin and will elute under the same salt conditions. RNase A, which is added at the beginning of the procedure, digests the liberated RNA efficiently during the alkaline lysis. The resulting RNA fragments do not bind to QIAGEN resin under the salt and pH conditions present in the lysate. The precipitated debris is removed by centrifugation or by use of a QIAfilter Cartridge, producing a cleared lysate for loading onto the QIAGEN-tip. It is important that the lysate is clear at this stage to ensure good flow rates and, ultimately, to obtain protein-free plasmid DNA preparations.

QIAfilter Cartridges are special filtration units designed to replace the centrifugation step after alkaline lysis of bacterial cells. After cultures are pelleted, bacterial cells are lysed in NaOH-SDS, neutralized by the addition of acidic potassium acetate, and incubated directly in the QIAfilter Cartridge. The lysate is cleared in a matter of seconds by passing the liquid through the filter. Insoluble complexes containing chromosomal DNA, salt, detergent, and proteins, which form during the neutralization step are completely removed. QIAfilter Cartridges clear bacterial lysates more efficiently than conventional centrifugation. In addition, small-sized SDS precipitates which cannot be separated by centrifugation are completely removed by using the QIAfilter Cartridge.
The cleared lysate is loaded onto a pre-equilibrated QIAGEN-tip by gravity flow. The salt and pH conditions of the lysate and the superior selectivity of the QIAGEN resin ensure that only plasmid DNA binds, while degraded RNA, cellular proteins, and metabolites are not retained and appear in the flow-through fraction. The QIAGEN-tip is then washed with medium-salt buffer (Buffer QC) which completely removes any remaining contaminants, such as traces of RNA and protein (e.g., RNase A), without affecting the binding of the plasmid DNA. Buffer QC also disrupts non specific interactions, and allows removal of nucleic acid-binding proteins without the use of phenol. The low concentration of alcohol in the wash buffer eliminates non specific hydrophobic interactions, further enhancing the purity of the bound DNA. The plasmid DNA is then efficiently eluted from the QIAGEN-tip with high salt buffer (Buffer QF or QN).
The eluted plasmid DNA is desalted and concentrated by isopropanol precipitation. Precipitation is carried out at room temperature to minimize coprecipitation of salt. After centrifugation, the DNA pellet is washed with 70% ethanol to remove residual salt and to replace the isopropanol with ethanol, which is more volatile and easily removed. The purified DNA is briefly air-dried and redissolved in a small volume of TE buffer, pH 8.0 or Tris•Cl, pH 8.5, and is ready for use in transfection, sequencing, labeling, cloning, or any other experimental procedure.
The eluted plasmid DNA is mixed with isopropanol and applied to the QIAprecipitator Module using the syringe provided in the kit. The module traps the precipitated DNA while the isopropanol mixture flows through. An additional ethanol wash step is recommended, to maximize DNA purity. The precipitated DNA is trapped in the QIAprecipitator as a thin layer, which allows thorough drying and removal of ethanol by simply pushing air through the QIAprecipitator with a syringe. The DNA is then eluted from the QIAprecipitator into a microcentrifuge tube with Buffer TE provided in the kit. Alternatively, any common buffer or water can be used. DNA should be stored at –20°C when eluted with water, as DNA may degrade in the absence of a buffering and a chelating agent. The DNA is ready for use in transfection, sequencing, labeling, cloning, or any other experimental procedure.