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

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

Features

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

Product Details

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.

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+
pUC18/19Lorist pBeloBAC DH5α
HB101
pBluescriptLawrist   
XL1-Blue
NM554
pGEMpWE15  SOLR  
pTZ19R pOU61 DH10B  
See figures

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.

See figures

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

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsAutomated sequencing, restriction digestion, microarray etc.
Plasmid typePlasmids, cosmids, BACs
Culture volume/starting material1.3 ml culture volume
Samples per run (throughput)1–4 x 96 samples per run
TechnologyQIAfilter technology
ProcessingManual (vacuum or centrifugation)
Yield<10 µg
Time per run or prep per run70 min/plate

Resources

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

Publications

Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells.
Lopez-Ochoa L; Ramirez-Prado J; Hanley-Bowdoin L;
J Virol; 2006; 80 (12):5841-53 2006 Jun PMID:16731923
Recombination in Thermotoga: implications for species concepts and biogeography.
Nesbø CL; Dlutek M; Doolittle WF;
Genetics; 2005; 172 (2):759-69 2005 Dec 1 PMID:16322518
Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.
Thomas R; Scott A; Langford CF; Fosmire SP; Jubala CM; Lorentzen TD; Hitte C; Karlsson EK; Kirkness E; Ostrander EA; Galibert F; Lindblad-Toh K; Modiano JF; Breen M;
Genome Res; 2005; 15 (12):1831-7 2005 Dec PMID:16339382
Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal.
Brauch KM; Dhruv ND; Hanse EA; Andrews MT;
Physiol Genomics; 2005; 23 (2):227-34 2005 Aug 2 PMID:16076930
The dominant inhibitory chalcone synthase allele C2-Idf (inhibitor diffuse) from Zea mays (L.) acts via an endogenous RNA silencing mechanism.
Della Vedova CB; Lorbiecke R; Kirsch H; Schulte MB; Scheets K; Borchert LM; Scheffler BE; Wienand U; Cone KC; Birchler JA;
Genetics; 2005; 170 (4):1989-2002 2005 Jun 14 PMID:15956664

FAQ

Can I use LyseBlue with R.E.A.L. Prep 96, QIAwell Ultra, or QIAprep 96 Turbo Miniprep Kits for the BioRobot systems?

For high-throughput BioRobot plasmid isolation systems such as R.E.A.L. Prep 96, QIAwell Ultra, or QIAprep 96 Turbo Miniprep, there is no need for visual lysis control with LyseBlue reagent, because

  1. insufficient resuspension and lysis have not been observed with these systems
  2. automation systems already handle the lysis steps well enough
  3. optical reading capabilities are not implemented on our BioRobot platforms
FAQ ID -863
Can Buffer R1 of the R.E.A.L. Prep 96 Plasmid Kit be replaced with Buffer P1?

Yes, it is possible to use Buffer P1 instead of Buffer R1 with the R.E.A.L. Prep 96 Plasmid Kit.

FAQ ID -823
Can BAC DNA prepared with the R.E.A.L. Prep 96 Plasmid Kit be used for high-throughput sequencing?

Yes. DNA prepared using the R.E.A.L. Prep 96 Plasmid Kit is well suited for high-throughput Capillary Electrophoresis (CE) DNA sequencing, or for fluorescent BAC DNA end sequencing on ABI PRISM 377 vertical slabgel sequencers. An optimized protocol for the purification of BAC DNA suitable for end sequencing on CE machines using R.E.A.L. Prep 96 is presented in QIAGENNews article 'High-throughput sequencing of plasmid and BAC DNA prepared using the REAL Prep 96 Plasmid Kit'. Typical sequence read lengths are around 600 bp.

High sample quality is essential for most sequencing applications on CE instruments. After intensive use of such sequencers (e.g., ABI PRISM 3700, MegaBACE 1000) in QIAGEN laboratories, a large set of statistical data on the quality and performance of DNA purified using the R.E.A.L. Prep 96 Plasmid Kit is available.

If you would like to take advantage of the Sequencing Service that we offer here at QIAGEN, please contact QIAGEN Technical Service.

FAQ ID -643