R.E.A.L. Prep 96 System

The Rapid Extraction Alkaline Lysis system allows economical, high-throughput isolation of plasmids, cosmids, BACs, PACs, and P1s using multiwell format from bacterial cultivation to purification. The DNA is suitable for use in routine sequencing and screening applications. (For an overview of QIAGEN miniprep products, see the Plasmid Miniprep Kit Selection Guide.)

Features and benefits

• Fast, high-throughput procedure
• 4 x 96 minipreps in 60–75 minutes
• Multiwell format from culture to DNA
• Optimized protocols for plasmids, cosmids, BACs, PACs, and P1s

Principle

The R.E.A.L. Prep 96 procedure processes multiples of 96 samples in parallel using both vacuum-driven transfer and centrifugation. The procedure is based on a modified alkaline lysis followed by efficient filtration through a special asymmetric 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.

The R.E.A.L. Prep 96 procedure is ideal 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").

Procedure

Cultures grown in multiwell blocks are harvested and lysed using a modified alkaline lysis procedure (see flowchart "R.E.A.L. Prep 96 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.

Applications

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:

• Automated fluorescent sequencing (see figures "Reliable Sequencing of Shotgun Clones" and "BAC End-Sequencing Using R.E.A.L. Prep 96 Kit"), including capillary sequencing, particularly when using improved thermostable polymerases (e.g., AmpliTaq DNA Polymerase, FS, or Thermo Sequenase)
• Typical screening procedures such as restriction digestion (see figure "Reproducible Yields") and microarray based analysis

Reliable Sequencing of Shotgun Clones

Phred 20 analysis of 11,808 plasmid shotgun sequences derived from a bacterial genome prepared with the R.E.A.L. Prep 96 System, were sequenced with BigDye Terminator sequencing chemistries (quarter reactions) and analyzed on an ABI PRISM 3700 DNA Analyzer with a POP-5 gel matrix. The analysis shows the percentage of sequencing reads, which were calculated using sequential windows of 10 bases.

BAC End-Sequencing Using R.E.A.L. Prep 96 Kit

Phred 20 analysis of BAC end-sequencing reactions from 8448 BAC clones, prepared using the modified R.E.A.L. Prep 96 procedure. The reactions were sequenced using BigDye Terminator sequencing chemistries (full reactions) and analyzed using an ABI PRISM 3700 DNA Analyzer with a POP-5 gel matrix. The analysis shows the percentage of sequencing reads, which were calculated using sequential windows of 10 bases.

Statistical Overview Number of reactions 16,750 Number of reactions of acceptable quality (>120 bases) 13,611 (81.3%) Number of reactions below acceptable quality (<120 bases) 3139 (18.7%) Success rate 81.3% Average read-length 432 bases

Reproducible Yields

Agarose gel analysis of shotgun clones from an S. pombe shotgun library purified using the R.E.A.L. Prep 96 procedure. Approximately 200 ng DNA was loaded per lane onto a 1% TAE agarose gel. Outer lanes: markers

Plasmids, cosmids, BACs, and bacterial strains used with R.E.A.L. Prep 96

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

For 1–4 x 96 rapid extraction alkaline lysis minipreps

	R.E.A.L. Prep 96 Plasmid Kit	R.E.A.L. Prep 96 BioRobot Kit
Format:	96-well plates	96-well plates
Lysate clearing:	QIAfilter 96 Plates	QIAfilter 96 Plates
Throughput:	1–4 x 96 samples	1–4 x 96 samples
Preparation time:	96 parallel minipreps in 60-75 minutes	96 parallel minipreps in 45 minutes
Equipment required:	QIAvac 96	BioRobot System

The R.E.A.L. Prep 96 Plasmid and BioRobot Kits provide QIAfilter 96 plates, growth blocks, and other components for preparing multiples of 96 plasmid or cosmid minipreps. The R.E.A.L. Prep 96 Kit uses the QIAvac 96, whereas the R.E.A.L. Prep 96 BioRobot Kit is a special kit format optimized for use with the BioRobot 3000. 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. 48-well blocks are not included in R.E.A.L. Prep 96 Plasmid or BioRobot Kits.

R.E.A.L. Prep 96 Handbook 02/02 (PDF version, 1164 KB)

Ordering Information Product Contents Cat. No. R.E.A.L. Prep 96 Plasmid Kit (4) For 4 x 96 rapid extraction alkaline lysis minipreps: 4 QIAfilter 96 Plates, Square-Well Blocks, Tape Pads, Reagents, Buffers 26171 R.E.A.L. Prep 96 Plasmid Kit (24) For 24 x 96 rapid extraction alkaline lysis minipreps: 24 QIAfilter 96 Plates, Square-Well Blocks, Tape Pads, Reagents, Buffers 26173 R.E.A.L. Prep 96 BioRobot Kit (4) For 4 x 96 rapid extraction alkaline lysis minipreps: 4 QIAfilter 96 Plates, Flat-Bottom Blocks and Lids, Square-Well Blocks, Reagents, Buffers, Tape Pads 961141 QIAvac 96 Vacuum manifold for processing QIAGEN 96-well plates: includes QIAvac 96 Top Plate, Base, Waste Tray, Plate Holder, Rack of Collection Microtubes (1.2 ml) 19504 Vacuum Regulator For use with QIAvac manifolds 19530 Square-Well Blocks (24) 96-well blocks with 2.2 ml wells, 24 per case 19573 48-Well Blocks (24) 48-well blocks with 5 ml wells, 24 per case 19577