This second generation plasmid mini prep kit yields 40-70 µg of high quality DNA in less than 30 minutes using the same format as the Plasmid Mini Kit I. The total DNA binding capacity is increased to allow culture volumes of up to 15 ml to be used, thus bridging the gap between the mini and midi prep protocols. Sufficient plasmid DNA can be readily and quickly isolated for dye-terminated fluorescent DNA sequencing and in vitro transfections. Only the Q-Spin column option is available for this kit.

Principle

The Plasmid Miniprep Kit II combines the power of HiBind™ technology with the time-tested consistency of alkaline-SDS lysis of bacterial cells to deliver high quality DNA in under 1 hour. HiBind™ matrix avidly, but reversibly, binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with de-ionized water or low salt buffer. 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

Pellet the bacterial cells from 10 - 15 ml overnight culture. Lyse with Solutions I and II and neutralize with Solution III. Pellet chromosomal DNA and cellular debris. Pass the cleared lysate through the column to bind plasmid DNA. Wash once with buffer HB and twice with DNA wash buffer. Dry the column. Elute plasmid DNA with de-ionized water or TE buffer for further use.

Applications

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

• Automated fluorescent DNA sequencing
• Restriction endonuclease digestion
• In vitro transfections

Agarose Gel analysis of the E.Z.N.A.^® Plasmid Miniprep protocol
by using 3 ml bacterial cultures. Lanes represent 10% of total
sample in each case. A: cleared bacterial lysate; B: eluted plasmid
DNA from column; M: Lambda Hind III Marker.

Sequencing analysis of the E.Z.N.A.^® Plasmid Miniprep II DNA Kit. Plasmid DNA was
purified using the E.Z.N.A.^® Plasmid Miniprep DNA Kit II, then sequenced and analyzed
on an ABI PRISM™ DNA Sequencer.