5 PRIME's new PerfectPrep mini system is, like most plasmid preps, available today based on two technologies. The first is the Maniatis alkaline-lysis method to release plasmid DNA from its bacterial hosts - leaving denatured protein and genomic DNA behind in a salt aggregation. Plasmid DNA in the alkaline lysis method was previously cleaned up by messy phenol/chloroform extraction and, when ultra-pure DNA was required, by tedious cesium chloride gradient centrifugation. These steps were replaced by the second technology: Nucleic acid-specific chromatography. With nucleic acid chromatography, intact RNA and DNA are bound to a bead matrix, usually acid washed glass beads or cretaceous earth (crushed diatoms), by a chaotropic salt like Guanadine HCl. Protein and lipids that weren't pelleted previously are washed off of the bead matrix by an ethanol salt solution. Because RNAseA is added in the first step, only DNA is left on the matrix. The pure DNA is then eluted in a low salt buffer, usually 50 mM TrisHCl, pH 8.0, or Tris/EDTA (TE buffer).
This method, when done carefully, produces plasmid DNA cleaner than cesium chloride centrifugation without the tedium and danger of cesium chloride or phenol/chloroform. What distinguishes the PerfectPrep system from other plasmid preps is the speed of use. The average miniprep requires 15-40 minutes for a single sample and about an hour when following manufacturers instructions (even Eppendorf's own older version of this product required 25 minutes). The new PerfectPrep system can process a sample in 5 minutes and 10 samples in under 20 minutes. Further, without the addition of specialized reagents, this system appears to work well with endA positive bacterial strains as well as with strains with high oligosaccharide content. I typically use 1.5 ml of DH5alpha culture with a high copy number plasmid (such as pBluescript) and obtain greater than 2.5 ug DNA. The A260/A280 ratio of the resulting plamid is usually at or near 1.8 and is suitable for all molecular biology applications.
This kit is quick, inexpensive, reproducible, and gives high yields of high quality DNA. It even has an innovative collection tube with a pinched bottom, which allows the researcher to clearly see small volumes of fluid. The only criticism that I have is that the kit requires similar but different volumes for the DNA binding (450 ul) and wash (400 ul) steps, while all three steps of the lysis require 100 ul volumes. This slight change has led to minor confusion on my part, but not a significant loss of performance. In summary, the 5 PRIME PerfectPrep Mini kit is an excellent product by almost every criterion.
Kevin L. Lorick, Ph.D.
Laboratory of Immune Cell Biology
National Cancer Institute, Division of Basic Sciences
At the time of this review the PerfectPrep Mini Kit was commercialized by Eppendorf. Today it is available from 5 PRIME. This review was edited to reflect this fact.