microR.E.A.L. Prep 384 Plasmid System

The microR.E.A.L. System allows economical very high-throughput isolation of bacterial plasmids from small-volume cultures. The DNA is highly suited for use in high-throughput automated sequencing. (For an overview of QIAGEN miniprep products, see the Plasmid Miniprep Kit Selection Guide.)

Features and benefits

• Simple, cost-effective method
• Up to 2 x 384 minipreps in 135 minutes
• Choice of multiwell culture formats

Principle

The microR.E.A.L. Prep 384 System processes multiples of 384 samples using centrifugation for rapid, very high throughput purification of plasmids from small-volume bacterial cultures. The procedure is based on a modified alkaline lysis procedure, which has been adapted for 384-well processing. Typically yields of 1–1.5 µg plasmid DNA are obtained from 300 µl (384-well block cultivation) or 350 µl (96-well cultivation) bacterial cultures grown in 2x YT medium.

Procedure

Cultivation and lysis can be carried out in either four 96-well cultivation plates or in 384-well blocks (UniPlate 400, available from Polyfiltronics Inc.). Lysates from either format are then transferred to a 384-well filter plate. Denatured and precipitated genomic DNA, proteins, and carbohydrates are efficiently removed by centrifugation of the 384-well filter plate. DNA in the cleared lysate is then concentrated by isopropanol precipitation.

Applications

The microR.E.A.L. Prep 384 procedure gives reproducible yields of DNA of an amount and quality suitable for routine high-throughput applications:

• Automated sequencing, on capillary and slab gel-based systems (see Figure "Reliable Sequencing Using the microR.E.A.L. Prep 384 Plasmid Kit").

Reliable Sequencing Using the microR.E.A.L. Prep 384 Plasmid Kit

Phred 20 analysis of bacterial clones purified using the microR.E.A.L. Prep 384 Plasmid Kit. Plasmid shotgun sequences (2323) were derived from a bacterial genome project. Clones were sequenced using BigDye Terminator chemistry and analyzed using an ABI PRISM 3700 DNA Analyzer with POP-5 gel matrix. The analysis shows the percentage of sequencing reads, which were calculated using sequential windows of 10 bases.

Reproducible Plasmid Purification

Agarose gel analysis of shotgun clones purified using the microR.E.A.L. Prep 384 procedure. 20% (6 µl of 30 µl) of the yielded plasmid DNA was loaded per lane. Markers: 100 ng plasmid.

microR.E.A.L. Prep 384 Plasmid Kit

For rapid extraction alkaline lysis minipreps in 384-well format

Format:	384-well plates
Lysate clearing:	Filtration
Throughput:	1–4 x 384 samples
Preparation time:	2 x 384 samples in 135 minutes
Equipment required:	Centrifuge

The microR.E.A.L. Prep 384 Kit provides 384-well filter plates, 384-well collection plates and all components required for preparing multiples of 384 plasmid minipreps using a centrifuge. 96-well microplates or 384-well blocks* used for cultivation of the bacterial clones are not included in the microR.E.A.L. Prep 384 Plasmid Kit.

Note: For growing bacterial cultures in 96-well format for the microR.E.A.L. Prep 384 procedure, 96-Well Microplates MC are required. For cultivation in 384-well blocks, 384-well blocks* and AirPoreTape Sheets are required.

microR.E.A.L. Prep 384 Plasmid Handbook 06/01 (PDF version, 308 KB)