R.E.A.L. Prep 96 Plasmid Kit

The Rapid Extraction Alkaline Lysis system allows economical, high-throughput isolation of plasmids, cosmids, BACs, PACs, and P1s using a multiwell format, from bacterial cultivation to purification. The R.E.A.L. Prep 96 Plasmid procedure achieves reproducible yields of plasmid DNA (see figure "Reproducible yields") suitable for routine high-throughput applications, including automated sequencing (see figure "Reliable sequencing of shotgun clones") and screening procedures, such as restriction digestion and microarray-based analysis.

The R.E.A.L. Prep 96 system provides a procedure suitable for use in high-throughput automated sequencing projects. A variety of high-copy plasmids, low-copy plasmids, BACs, and E. coli strains have all been used successfully (see table "Plasmids, cosmids, BACs, and bacterial strains used with R.E.A.L. Prep 96").

 The R.E.A.L. Prep 96 procedure is based on modified alkaline lysis of bacterial cells, followed by clearing of the lysates by filtration using the QIAfilter module, and further concentration of DNA by isopropanol precipitation. The DNA obtained is resuspended in a small volume of Tris buffer and is ready for use. All steps are performed without the use of phenol, chloroform, CsCl, and ethidium bromide.

The R.E.A.L. Prep 96 procedure processes multiples of 96 samples in parallel using both vacuum-driven transfer and centrifugation (see flowchart "R.E.A.L. Prep 96 procedure"). The procedure is based on a modified alkaline lysis followed by efficient filtration through a special filter unit, QIAfilter 96. Typically, yields of up to 7 µg plasmid DNA are obtained from 1.3 ml LB cultures or up to 20 µg with TB or similar super-rich media. With the optimized BAC protocol, up to 800 ng large-plasmid DNA can be isolated from 2.5 ml bacterial cultures.

Cultures grown in multiwell blocks are harvested and lysed using a modified alkaline lysis procedure. An optional heating step further denatures and precipitates proteins and carbohydrates, which are then removed by vacuum filtration through the QIAfilter 96 plate. DNA in the filtrates is concentrated by isopropanol precipitation.

The procedure functions optimally when the same vector–host combination is used for all samples in a given block, creating standardized conditions. Different vector–host strain combinations should be optimized individually with respect to medium selection and culturing time.

The R.E.A.L. Prep 96 Plasmid Kit provides QIAfilter 96 plates, growth blocks, and other components for preparing multiples of 96 plasmid or cosmid minipreps. The R.E.A.L. Prep 96 Plasmid Kit uses the QIAvac 96. BAC purification using the R.E.A.L. Prep 96 procedure requires the use of 48-well blocks to provide optimal culture conditions for the BAC clones. The 48-well blocks are not included in the R.E.A.L. Prep 96 Plasmid Kit. Parallel processing allows 96 samples to be purified in 60–75 minutes.

The R.E.A.L. Prep 96 procedure gives reproducible yields of DNA of an amount and quality suitable for many routine, high-throughput applications:

• Sequencing
• Screening procedures