The only maxi-scale system to deliver high quality plasmid DNA using the spin-column format - without costly accessories. Up to 250-500 ml of overnight culture (log phase) in LB medium typically produces 500-1000 µg high-copy plasmid DNA and up to 1 liter of cell culture (log phase) may be processed when working with low-copy number plasmids.

Principle

The Plasmid Maxiprep Kit combines the power of HiBind^® technology with the time tested consistency of alkaline-SDS lysis of bacterial cells to deliver high quality plasmid DNA. The maxi-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. HiBind^® matrix that avidly, but reversibly, binds DNA under specific conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with de-ionized water or low salt buffer.

Procedure

Pellet cells from approximately 500 ml of bacterial culture. Re-suspend collected cells in Solution I / RNase I. Add Solution II and gently mix by inverting and rotating the tube to obtain a cleared lysate. Addition of Solution III neutralizes the reaction, enabling the DNA to bind to the HiBind^® column . Pellet chromosomal DNA and cellular debris, by a brief centrifugation step, and pass the cleared lysate through the column to bind plasmid DNA. Bound DNA is subjected to multiple wash steps (once with HB buffer and twice with wash buffer) and the column is dried well prior to elution with de-ionized water or TE buffer.

Applications

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

• Automated fluorescent DNA sequencing
• Restriction digestion
• Transfection of mammalian cells
• In vitro transcription

1% agarose gel analysis of the E.Z.N.A.^® Plasmid Maxiprep Kit (D6922) using
500 ml overnight culture of LB medium incubated DH5a harboring PCU 19 plasmid.
Lanes represent 0.1% of the total sample in each case. A: cleared bacterial
lysate; B: eluted plasmid DNA from column; M: Lambda Hind III marker.

Dye terminator sequencing of plasmid DNA isolated with the E.Z.N.A.^® Plasmid
Maxiprep Kit (D6922) and sequenced using AmpliTaq^® DNA Polymerase.
Performed on an ABI Prism™ 377 Version2.1.1 Sequencer.