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DirectPrep 96 Miniprep System for cost-effective, high-throughput plasmid ,,, DNA purification

QIAGEN introduces the DirectPrep 96 Miniprep System for cost-effective, fully automated, high-throughput DNA purification from high-copy plasmids. Use of a novel lysis chemistry eliminates the need for a lysate clearing step. Plasmid DNA purification is performed using a single plate and a simple bindwashelute procedure. The protocol is faster and easier to perform than existing two-plate methods. The DirectPrep 96 procedure is designed for use on BioRobot 3000 or BioRobot 8000 workstations, where 4 plates can be processed in parallel, and can be performed manually using the QIAvac Multiwell. The DirectPrep 96 Miniprep System yields plasmid DNA suitable for all routine applications, including automated fluorescent sequencing, restriction digestion, and PCR.

The DirectPrep 96 Miniprep System provides:

Economy for high-throughput sequencing and screening projects
Easy handling simple one-plate procedure
Flexibility automated or manual procedures available
Speed 96 samples in just 35 minutes (manual procedure)
SBS (Society for Biomolecular Screening) plate standard easy to automate

Flexible procedure DirectPrep 96 Procedure The DirectPrep 96 Miniprep method is a simple one-plate procedure. All lysis steps are carried out in the same 96-well flat-bottom block used for growth of the bacteria. After alkaline lysis of the pelleted cells, the lysate is neutralized using an optimized buffer, which leads to a virtually clear lysate. Timeconsuming centrifugation or filtration of the lysate is not required, a nd chaotropic salts are not used in the procedure. Subsequent addition of isopropanol provides optimal binding conditions for the plasmid DNA to the DirectPrep 96 membrane. Lysates are then transferred to the DirectPrep 96 Plate where all subsequent steps take place (see flowchart). After washing, the membrane is dried and pure plasmid DNA is eluted. The DirectPrep 96 System yields ready-to-use plasmid DNA that is highly suited for all standard downstream applications. Reproducible yield and purity

Plasmid DNA yields depend on the culture conditions and the hostvector system. Using the DirectPrep 96 System, yields of up to 4 g high-copy plasmid are typically obtained from 1.25 ml E. coli culture grown in LB medium. An average DNA concentration of >50 ng/l is obtained in an eluate volume of 50 l (Figure 1). The purified DNA shows an average A260/A280 ratio of 1.82 (Figure 2), and plasmid DNA is immediately ready to use in sequencing reactions and other downstream applications. Overall, the yield of plasmid DNA is sufficient for several sequencing reactions and additional archiving of the sample.

Reproducible Plasmid Purification Using DirectPrep 96 Miniprep Kits Reproducible DNA Purity Using DirectPrep 96 Miniprep Kits Figure 1 Agarose gel analysis of shotgun clones. A pUC19-based shotgun library transformed into E. coli strain DH10B was purified using the DirectPrep 96 Miniprep Kit. Cultures were grown in 1.25 ml LB medium for 20 hours. 3 l of a 50 l eluate of the purified plasmid was loaded per lane. M: 150 ng 3 kb marker plasmid DNA. Figure 2 E. coli strain DH10B was transformed with plasmid pUC19 and grown in a flat-bottom block containing 1.25 ml LB medium for 20 hours. Plasmid DNA was purified using the DirectPrep 96 Miniprep Kit. Eluted plasmid DNA was diluted in Buffer EB and the A260/A280 ratio was measured. An average ratio of 1.82 was obtained. DNA for high-throughput applications

The plasmid DNA obtained using the DirectPrep 96 Miniprep System is highly suited for automated sequencing applications, such as high-throughput fluorescent sequencing, restriction enzyme digestion, or PCR. Results of high-throughput capillary sequencing of plasmid DNA purified using the DirectPrep 96 Miniprep Kit are shown in Figures 3 and 4. DNA isolated using the DirectPrep 96 procedure shows a typical read-length of 600 bases when used in capillary sequencing (Figure 3). Using either BigDye terminator v.2.0 or v.3.0 sequencing chemistry, the average read-length obtained with DirectPrep 96 purified DNA is in the same range as silicabased plasmid purification methods (Figure 4).

Phred Analysis of Bacterial Clones Prepared Using the DirectPrep 96 Miniprep Kit Figure 3 Phred 20 analysis of sequencing reactions. Plasmid pUC19 was purified using the DirectPrep 96 Miniprep Kit and reactions were sequenced using BigDye terminator v. 2.0 chemistry and analyzed using an ABI PRISM 3700 DNA Analyzer with POP-5 gel matrix. The analysis shows the perc entage of sequencing reads at each read-length, which was calculated using sequential windows of 10 bases. High-Quality DNA for Automated Sequencing Figure 4 Typical fluorescent capillary sequencing result obtained with plasmid DNA purified using the DirectPrep 96 Miniprep Kit. Plasmid pUC19 was sequenced using BigDye terminator v. 3.0 chemistry in a cycle sequencing reaction on an ABI PRISM 3700 DNA Analyzer. The sequencing reaction was purified using the DyeEx 96 Kit (QIAGEN, cat. no. 63181). Conclusions
  • The novel lysis chemistry of the DirectPrep 96 Miniprep System enables a one-plate method for simple, rapid, and cost-effective high-throughput plasmid preparation.
  • The system yields DNA of an amount and quality suitable for all standard downstream applications.

* Requires use of BioRobot systems or the QIAvac Multiwell.
Larger kit sizes available; please inquire.



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