Qiagen produces an excellent range of products for the isolation and purification of nucleic acids in various contexts. Recently, they have added to this line-up with the release of the HiSpeed Plasmid Kit. This kit shares many of the common Qiagen themes, but radically reduces the time for a plasmid prep to below one hour. Earlier Qiagen plasmid preparation kits consisted of centrifugation steps that alone accounted for much more time.
Nucleic acid purification products from Qiagen are based upon patented ion-exchange resins with positively charged DEAE groups that interact with the phosphate groups of nucleic acid backbones. The resins are made of silica beads with a large pore size, this results in a large surface area and exceptionally high charge density. Qiagen resins eliminate the use of toxic chemicals (such as phenol and ethidium bromide) and expensive equipment (ultracentrifuges), instead relying upon simple chromatography and common lab apparatus such as microfuges. Qiagen products with such resins have been applied to the isolation of pcr products, plasmids, cosmids, electrophoresed DNA bands in agarose and RNA from various tissues.
For plasmid preparations, both the new HiSpeed kit and the older Qiagen kits rely upon traditional alkaline lysis (200 mM NaOH, 1 % SDS) to disrupt cells. The detergent solubilizes the cell membrane to release the cell contents and the alkaline conditions denature both plasmid and chromosomal DNA and proteins. The lysate is then neutralized and ionic strength increased with potassium acetate. These conditions cause SDS to precipitate and form complexes with denatured proteins, chromosomal DNA and cell fragments. The two strands of plasmid DNA are, however, in close proximity because they are covalently closed and so renature to form soluble double stranded DNA. Exposure of bacterial cells to alkaline conditions for longer than the suggested time has two consequences: 'too much' cellular disruption with the release of large amounts of chromosomal DNA and excessive denaturation of plasmid DNA, both these events contaminate plasmid preparations.
After cell lysis, the HiSpeed kit departs from the centrifugation based kit that I have used previously (but maybe not too much from kits relying upon filters for lysate clarification). Instead of incubation on ice for 15 mins to allow SDS precipitation to occur, the neutralized lysate is transferred to a first syringe type device, the QIAfilter. The end of the QIAfilter is initially capped to prevent flow and the lysate incubates at room temperature for 10 minutes. During this time the precipitants accumulate at the top of the capped syringe as a fluffy white layer. The cap is then removed and the plasmid containing solution, minus the precipitate which is filtered out, is expelled from the QIAfilter into a pre-equilibrated 'HiSpeed tip' which resembles Qiagen's older 'tips' that contain the resin. The HiSpeed tip seems to flow much faster (> 1 ml/min) than older Qiagen tips, perhaps because lysates are 'cleared' more efficiently. I have previously relied upon 45 minutes of centrifugation to clear lysates prior to tip loading, so these procedures constitute a substantial time savings. Once the cleared lysate is loaded, the column is washed with 20 ml of a 'medium salt' wash buffer to remove remaining contaminants. The washed plasmid DNA is then eluted from the HiSpeed tip with a 'high salt' elution buffer and precipitated with isopropanol at room temperature which minimizes salt co-precipitation.
From the isopropanol precipitation step onward the HiSpeed kit radically departs from all older Qiagen kits and even more time is saved. Using a 20 ml syringe, the precipitated DNA is passed through another filter, the QIAprecipitator, which collects precipitated DNA. The QIAprecipitator resembles a 0.22 or 0.45 mm syringe filter and is easily added to or removed from the syringes used at this stage of the prep. Residual isopropanol is removed from the QIAprecipitator by using the 20 ml syringe to pass air over the unit and by blotting with tissue paper. A final 5 ml syringe is then used to apply TE buffer to the QIAprecipitator so that plasmid DNA is eluted. These last procedures take less than 5 minutes to complete.
The advantages of this product are clear in terms of the time saved in plasmid preparation. What used to 'effectively' be an all day procedure is now something that can be performed in just under one hour. A product that greatly reduces the time for a procedure is only really beneficial, however, if it is also cost effective and yields and quality are comparable. In terms of cost, HiSpeed preps are around $1.50 more expensive per prep than the older Qiagen kit that I used. In terms of the quality of the plasmid produced, I had a harvested culture in two portions stored in the freezer (200 ml between two centrifuge pots). The QIAprecipitator kit produced twice the plasmid, of equal purity, as compared to the older centrifuge based kit. This difference probably reflects loss of plasmid during the precipitation steps performed in a centrifuge with the older kit. That said, if I don't need to account for such a problem with the QIAprecipitator, then the new kit is more reliable and yields are effectively greater.
Peter Haggie, PhD
Post-Doctoral Fellow
University of California, San Francisco