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

For rapid purification of sequencing-grade plasmid, cosmid, BAC, PAC, or P1 DNA

  • Optimized plasmid, cosmid, BAC, PAC, and P1 protocols
  • Highest possible DNA yields

R.E.A.L. Prep 96 Kits provide 96-well purification plates suitable for processing on the QIAvac 96 or BioRobot 3000 and BioRobot 8000 workstations, yielding sequencing-grade plasmid DNA.
For optimal results it is recommended to use this product together with QIAvac 96.

Cat No./ID: 26173
R.E.A.L. Prep 96 Plasmid Kit (24)
€4,430.00
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For 24 x 96 rapid extraction alkaline lysis minipreps: 24 QIAfilter 96 Plates, Square-Well Blocks, Tape Pads, Reagents, Buffers
The R.E.A.L. Prep 96 Plasmid Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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R.E.A.L. Prep 96 procedure.
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 filter unit, the QIAfilter 96.
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Reproducible yields.
Agarose gel analysis of shotgun clones from an S. pombe shotgun library purified using the R.E.A.L. Prep 96 procedure is shown. Approximately 200 ng DNA was loaded per lane onto a 1% TAE agarose gel. Outer lanes: markers
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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 is shown. Samples 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.
Performance

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.

Plasmids, cosmids, BACs, and bacterial strains used with R.E.A.L. Prep 96
Plasmids  Cosmids (45–50 kb)  BACs (80–120 kb) Bacterial strains
 endAendA+
pUC18/19 Lorist pBeloBAC DH5α
HB101
pBluescript Lawrist    
XL1-Blue
NM554
pGEM pWE15   SOLR  
pTZ19R  pOU61   DH10B  
Principle

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.

Procedure

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 6075 minutes.

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:

Sequencing
Screening procedures
Features
Specifications
Applications Automated sequencing, restriction digestion, microarray etc.
Culture volume/starting material 1.3 ml culture volume
Plasmid type Plasmids, cosmids, BACs
Processing Manual (vacuum or centrifugation)
Samples per run; throughput 1–4 x 96 samples per run
Technology QIAfilter technology
Time per run or prep per run 70 min/plate
Yield <10 µg

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Kit Handbooks (1)
For Rapid Extraction Alkaline Lysis Minipreps of plasmid, cosmid, or BAC DNA
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