Low yield or degradation are common occurrences in many large-scale plasmid preparations even when high- copy-number plasmids are used. In many cases, plasmids can be degraded after long term storage. Endonucleases are the major factors which cause this kind low yield or degradation. OBI's third generation HP Plasmid extraction system is designed to isolate high purity plasmid from bacterial hosts with elevated levels of endonuclease activity. In addition to improving plasmid DNA yield and purity, this system reduces overall protein contamination. This system is especially suitable for use with thawed plasmids that have been frozen for long term storage.

Principle

The High Performance Plasmid Midiprep and Maxiprep Kits combine the power of HiBind™ technology with the time-tested consistency of alkaline-SDS lysis of bacterial cells to deliver high quality DNA. HiBind™ matrix avidly, but reversibly, binds DNA under specific conditions allowing proteins and other contaminants to be removed by various wash steps. Nucleic acids are easily eluted with de-ionized water or low salt buffer. The High Performance Plasmid isolation Kit integrates OBI’s protease removal procedure into the standard E.Z,N.A.^® Plasmid isolation Kit. Plasmid isolated using this system has increased stability resulting in longer term storage. The use of midi-columns and maxi-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. For more information on columns, click Flexible Multi-Format Columns.

Procedure

Pellet bacterial cells from 50ml / 500ml of overnight culture. Re-suspend collected cells in Solution I / RNase A. Add Solution II and OB Protease mixture and gently mix by inverting and rotating the tube to obtain a cleared lysate. Incubate 15-20 minutes at room temperature. Add Solution III and gently mix by inverting several times until a flocculent white precipitate forms. Centrifuge to pellet the cellular debris and genomic DNA. Add the clear supernatant to a clean HiBind^® DNA midi / maxi column and centrifuge to completely pass the lysate through the column. Bound DNA is subjected to multiple wash steps with HB buffer, DNA wash buffer and absolute ethanol and dried well prior to elution with de-ionized water or TE buffer.

Applications

High Performance Plasmid Midiprep and Maxiprep Kits provide high quality, reproducible plasmid DNA for use in a number of applications including :

• Transfection of Mammalian cells
• Restriction Endonuclease Digestion
• Automated fluorescent DNA sequencing