Propel Your Research Using Proprep ™ For High-Throughput BAC & Plasmid DNA Isolation and Bioprocessing.

Propel Your Research Using Proprep ™ For High-Throughput BAC & Plasmid DNA Isolation and Bioprocessing.

Biotech Support Group’s Proprep ™ offers the ability to isolate DNA from recombinant DNA plasmids/bacteriophage or chromosomal/genomic DNA from prokaryotic or eukaryotic organisms. Proprep™ is a complete system for DNA isolation. The kit consists of Procipitate™, buffers and necessary hardware. The ProPrep™ strategy is to use Procipitate™ which binds proteins and other contaminants(>95%), DNA voids in flow-through & provides high quality DNA suitable for automated sequencing, southern blotting, and restriction digestion. Thus, it improves yield of DNA over alternative bind and elute systems by removing only the contaminants and leaving the DNA alone. This minimizes shearing, improves yield, and makes ProPrep™ ideal for BAC template preparation. Routine applications such as polymerase chain reaction (PCR) amplification, DNA sequencing, high throughput genomic screening and subcloning of transgenes are compatible with Proprep™.

After using Proprep the result is DNA which is free of contaminants including lipids, protein, carbohydrate, or other nucleic acid, for example, DNA free of RNA or RNA free of DNA. Moreover, the quality and integrity of the isolated DNA is preserved. The Proprep™ method offers a low cost, efficient, 96 well procedure for BAC end sequencing and works with high throughput methods for BAC end sequencing of all the clones. Overall, Proprep™ permits the selective isolation of plasmid DNA that can be used for transformation, DNA cloning experiments, in nick translation and for restriction endonuclease research.

The Proprep ™ method makes it easy to do analysis of clones by gel electrophoresis and yields plasmid DNA that is pure, digestible by restriction enzymes. The mechanism of Proprep ™ is selective alkaline denaturation of high molecular weight chromosomal DNA while keeping covalently closed circular DNA double-stranded. Adequate pH control and neutralization renatures the chromosomal DNA to form an insoluble clot while extracting the plasmid DNA in the supernatant. Using this method, both large and small plasmid DNA are efficiently extracted.

Perfect for screening clones from a BAC library plate, gene discovery and BAC end sequencing, the Proprep™ method ensures that the plasmid DNA is recovered from the supernatant after the denatured material has been removed by centrifugation.

1. Optimum mixing, temperature and method: Proper mixing is imperative to insure consistent results of Proprep™. Grow 1.5 – 2.0 ml overnight LB or 2xYT culture (for BACs 20 ug/ml chloramphenicol) to early stationary phase, a concentration range from 24x10 corresponds to OD600 (1:10 dilution) = 0.2 to 0.4. 2xYT broth provides approximately 50% greater DNA yield than LB. TB broth is not recommended.
2. Buffer: The buffer condition affects the sensitivity of proteins toward environmental pH changes. Proprep ™ offers a TE1 Resuspension Buffer. Add 120 ul of TE1 and 9 ul RNase cocktail to each well. Resuspend cells by vortexing. Insure that no cell clumps remain or yield will be reduced.
3. Water purity: Unpurified water contains a lot of microorganisms or proteases that will result in protein degradation.

Proprep™ is also ideal for bioprocessing which requires large-scale production of pDNA. As the industry shifts from small-scale plasmid production for cell transfection to large-scale production, the Proprep™ method is simple and effective for plasmid DNA because Procipitate™ binds to proteins and other contaminants(>95%), DNA voids in flow-through & provides high quality DNA suitable for automated sequencing, southern blotting, and restriction digestion. Thus, it improves yield of DNA over alternative bind and elute systems by removing only the contaminants and leaving the DNA alone.

References:

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