Omega'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. The HP Plasmid Miniprep Kit I allows isolation of up to 20 µg high quality plasmid from 1-5 ml endA+ bacterial cultures. HP Plasmid Miniprep II kit allows isolation of up 70 µg high quality plasmid from 10-15 ml endA+ bacterial cultures.

Principle

The Plasmid Miniprep Kits combine the power of HiBind™ technology with the time-tested consistency of alkaline-SDS lysis of bacterial cells to deliver high quality DNA in under 20 minutes. 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. Miniprep Kit I is simple, efficient, and fast to facilitate screening of recombinant clones. Miniprep Kit II employs an additional wash step that produces high quality DNA suitable for automated fluorescent DNA sequencing, restriction endonuclease digestion, transfection of mammalian cells, and other manipulations. Plasmid isolated using this system has increased stability resulting in longer term storage. The use of mini-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

Collect bacterial cells via a brief centrifugation step, and re-suspend in Solution I. Re-suspended cells are efficiently lysed via the addition of Solutions II and RNase A. Once lysis is complete Solution III is added to the mixture to neutralize the solution enabling efficient binding to the column. Prior to addition of the solution to the column a brief centrifugation step is carried out to pellet chromosomal DNA and cellular debris. The cleared lysate is subsequently added to the column to bind plasmid DNA. Bound DNA is subjected to multiple wash steps prior to elution with de-ionized water or TE buffer.

Applications

HP Plasmid DNA Kit provides high quality, reproducible plasmid DNA for use in the following applications :

• Eukaryotic and bacterial transformation
• Transfection of Mammalian cells
• DNA Modification
• Automated fluorescent DNA sequencing
• In vitro transcription
• Structural and Functional analysis of DNA
• Experimental genetic therapy

Gel Analysis of HP Plasmid Miniprep Kit II.
Plasmid DNA isolated from two endoA+ host
strains were run into 1% TAE agarose gel.
M: Lamda Hind III.