Preparation of the cell lysate
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.