EZ96 Fastfilter Plasmid Kit

Introduction

By integrating the E-Z 96^® Fastfilter plate with E-Z 96^® Plasmid Isolation Kit, this protocol provides a simple and reliable method for isolating plasmid DNA with a 96-well format. The entire procedure can be completed in less than 60 minutes and the purified DNA can be used directly for automated fluorescent sequencing and other standard downstream applications.

Principle

The E-Z 96^® Fastfilter Plasmid Purification 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. By using the E-Z 96^® DNA Plate, up to 96 samples can be simultaneously processed in less than 90 minutes. HiBind^® matrix that avidly, but reversibly, binds DNA 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 new E-Z 96^® Lysate Clearance Plate obviates time-consuming centrifugation for clearing of the bacterial alkaline lysates. It also has an average DNA recovery rate 10 to 30% higher than the manual centrifuge method.

Procedure

Bacterial cultures are grown in 1 ml LB medium containing an appropriate antibiotic. A 1 ml aliquot is transferred to a 96 well block or an individual 1.5ml centrifuge tube. After a brief centrifugation step, the cell pellet will be re-suspended with 220 μl of Solution I. Next, 220 μl of Solution II is added and kept at room temperature for 5 minutes. Add neutralization buffer and mix by gently shaking and rotating the plate for 1 minute until a flocculent white precipitate forms. Add GBT Buffer and gently mix by shaking and rotating the plate for 1 minute. Immediately transfer the lysate into the Lysate Clarence Plate. Allow the cell lysate to sit and pass through the filter using a vacuum manifold. Add 500 μl of HB Buffer to each well, apply the vacuum until all the liquid pass through. Wash the E-Z 96^® DNA Plate twice with DNA wash buffer and once with absolute ethanol. Remove the E-Z 96^® DNA plate from vacuum manifold, dry it well and elute the purified plasmid DNA with TE buffer or water using the vacuum manifold.

Applications

The E-Z 96^® Fastfilter Plasmid Isolation Kit provides high quality, reproducible plasmid DNA for use in a number of downstream applications including :

Automated fluorescent DNA sequencing