All of Qiagen’s plasmid purification kits are based on a modified alkaline lysis protocol, followed by the binding of DNA to an anion-exchange resin at low salt and pH conditions. Impurities are removed by a medium salt wash and plasmid DNA is eluted with a high salt buffer. Plasmid DNA is then concentrated and desalted with isopropanol precipitation. The beauty of these kits is that no major equipment is required and many of the toxic reagents associated with traditional methods of plasmid DNA purification, such as phenol, chloroform and ethidium bromide, are not used. The Qiagen HiSpeed Plasmid Kit comes in midi and maxi formats. My experience has been with midi kit, although the maxi kit protocol is essentially identical. Midi kits have a maximum binding capacity of 200 ug and the maxi 750 ug. All necessary reagents are provided with the kit, except isopropanol. The only equipment required are two bench top centrifuges; one to harvest the bacteria and the other a micro-centrifuge for Eppendorf tubes.
The protocol begins with the harvesting of the bacteria. I generally do this is an Eppendorf 5810R benchtop centrifuge using 50 ml falcons. I spin at 4000 rpm for 20 min (a lower speed than in the Qiagen protocol) following by resuspension of the bacteria in buffer P1 (Tris-EDTA buffer) and lysis in buffer P2 (200 mM NaOH; 1%SDS). This protocol serves to disrupt the cell membrane and allow the alkaline buffer to denature proteins and DNA. Addition of buffer P3 (3 M potassium acetate) causes the SDS to precipitate, taking chromosomal DNA, proteins and general cell debris, down with it. Plasmid DNA, being smaller and covalently bound, tends to re-nature correctly and forms soluble double-stranded DNA. It is important not to exceed the 5 minute incubation time recommended by Qiagen for the buffer P2 lysis step, as this can result in excessive denaturation of plasmid DNA. I usually finish this incubation in 2-3 minutes to prevent contamination with denatured plasmid DNA (although this might results in a lower overall DNA yield).
After these initial steps, the QIAfilter cartridge effectively removes the need for the high speed centrifugation step common to many other plasmid prep kits. The entire contents of the alkaline lysate are poured into the cartridge (closed at one end) and left for 10 minutes (allows precipitate to float to the top of the solution). A plunger is then inserted into the cartridge (which has been opened), pushed down and the cleared filtrate is applied to the pre-equilibriated HiSpeed tip. The precipitate should now be at the top of the solution, thus, it does not block the filter. The cleared lysate enters the anion-exchange resin in the HiSpeed tip by gravity flow. The resin is then washed with a medium salt buffer to remove contaminants, such as RNA and proteins. The bound plasmid DNA is then eluted with the high salt buffer and the plasmid DNA is precipitated by the addition of isopropanol. After incubation, this solution is run through the QIAprecipitator module that traps precipitated DNA, again removing the need for high speed centrifugation. The QIAprecipitator module is further washed with 70% ethanol and DNA is eluted by running a buffer (TE is provided) or water through the module. I normally elute from the QIAprecipitator with 0.5 ml water, giving a higher concentration than eluting with 1ml (useful for subsequent sequencing reactions and removes the need for sample concentration). At this point, the DNA is ready for further analysis (i.e. sequencing, site directed mutagenesis, transfection etc.).
This kit is fast, reliable and produces high yields of plasmid DNA of a quality and purity that meets the demands of most downstream applications, such as sequencing and transfections.
Boyd Scott
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals