Product Description
The NeXprep Ultra Pure Plasmid Purification Kits rapidly isolates and purifies covalently closed circular DNA such as BAC, PAC, fosmid, cosmid and plasmid from bacterial cells. The method comprises the steps of growth of microorganism, lysis of cells, separation of the target DNA from the cell debris, unwanted chromosomal DNA and proteins, and binding of the target DNA efficiently to a solid matrix under conditions suitable for the removal of insoluble materials. The process is conducted in the absence of centrifugation step (used for separating the target macromolecules from the un-wanted waste) and clear lysate transferring step (performs lysis and separation steps in a single plate. The binding of target DNA, removal of un-wanted waster and releasing of the target DNA are conducted in a second plate.
Accessories of the NeXprep Ultra Pure Plasmid Purification Kit include the DuraPoint Plate Piercer that converts the NeXplate from a culture plate to a filter bottom plate in seconds. It also converts the NeXbinding plate to DNA binding/releasing plate, by which un-wanted chromosomal DNA, proteins and cell debris can be easily to be removed, whereas, the high quality target DNA can produced. The DuraVac, a durable two-piece (top and bottom) manifold for the processing of either NeXprep Plasmid Purification or NeXprep Ultra Pure Plasmid Purification kit. A convenient pressure control valve provides the ability to adjust the pressure as required simply by turning the valve clockwise to increase the pressure and counter clockwise to reduce pressure. The DuraVac manifold allows you to perform NeXprep Plasmid and/or NeXprep Ultra Pure Plasmid purification utilizing any available vacuum source and allows you to collect DNA samples into any storage plate. Application: This system is streamlined so that a 96-well plate can be processed in approximately thirty (30) minutes providing ultra purified plasmid DNA from E. coli.
Downstream Application Suitability
For a multitude of uses including cloning, BAC-end sequencing, transformation, transfection, and restriction digestion.
Features
Process in high throughput (96-well) or low throughput (single column) format
Save hands-on time by eliminating clear lysate transferring or centrifugation steps
Microbial cell growth and cell lysis in the same plate
Suitable for isolating large construct vectors including BAC and PAC
Provides pure DNA for reproducible results in any downstream application
Process Overview
High Quality, Ultra Pure Plasmid DNA Preparation System
The DeWalch NeXprep Ultra Pure Plasmid DNA Purification Systems can be used to isolate ultra pure DNA of BAC, PAC, fosmid, cosmid and plasmid. The NeXprep Kit is an easy to use kit. Typically, approximately 2 µg of highly pure BAC DNA can be obtained form a 1.2-ml overnight bacterial cultures. The combination of DeWalch’s piercing technology and vacuum filtration make the kit convenient to use. Excellent sequencing results have been obtained using templates from BAC libraries of different organisms.
Examples of high quality BAC, PAC, fosmid, cosmid and plasmid purified using NeXprep Ultra Pure Plasmid DNA Purification Kits are shown in the following figures.
Fig. 1. Agarose gel (0.8%, Inversion electrophoresis) analysis of BAC DNA isolated from a BAC library. Eight (8) µl each of BAC DNA or a mixture of bacterial genomic DNA and plasmid pUC19 (M, outsider lanes) was electrophorezed for 90 min at 6 V/cm.
Fig. 2. Agarose gel (1%) analysis of fosmid DNA isolated from a fosmid library. Five (5) µl each of fosmid DNA or a mixture of bacterial genomic DNA and plasmid pUC19 (M, far right lane) were electrophorezed for 90 min at 6 V/cm.
Fig. 3. Agarose gel (1%) analysis of cosmid DNA isolated from a cosmid library. Five (5) µl each of cosmid DNA) was electrophorezed for 90 min at 6 V/cm.
Fig. 4. Agarose gel (1%) analysis of plasmid pSMART DNA isolated from a shotgun library. Five (5) µl each of plasmid DNA was electrophorezed for 90 min at 6 V/cm. A mixture of bacterial genomic DNA and plasmid pUC18 (far right lane) was also loaded on the gel as molecular markers.
An example of BAC-end sequencing electropherogram with ABI 3130 XL of the BAC prepared using the DeWalch NeXprep Ultra Pure Kit is shown in Fig. 5.
Fig. 5. ABI 3130xl Genetic Analyzer automated fluorescent DNA cycle sequencing of a Xenopus BAC clone purified using DeWalch’s NeXprep Ultra Pure Plasmid Kit. Using ABI KB basecaller software (ABI DNA sequencing analysis software V5.2), the QV20 score of this particular sequences is 961 bases.
Example of BAC-end sequencing distribution from a human BAC library is shown in Figure 7. The average QV20 score of this plate sequencing was 765 bases with a pass rate of 98%.
Fig. 6. Sequencing results of BAC clones using DNA purified using the DeWalch NeXprep Ultra Pure Plasmid Purification Kit. DNA sequencing was performed using AB (Applied Biosystems) Big Dye Terminator v3.1 Cycle Sequencing Kit each with a 1/8 X reaction. Data was analyzed using AB DNA Sequencing Analysis Software V5.2.
DeWalch NeXprep Plasmid Purification Kits Ordering Information
To place an order or need a Technical Assistance, call 800-880-8993 or e-mail us at www.dewalch.com/NeXprep.
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