Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

An Efficient Method For The Purification Of Amplified, Large Insert DNA Clones.

Patrick J. O'Mullan

  Princeton Separations, Inc. PO Box 300 Adelphia, NJ 07710

We have developed a quick and reliable method for purifying large DNA clones (amplified to high copy number) such as fosmids and BACs. The system uses a modification of the PSIClone BAC DNA kit involving a vacuum manifold adapter which can fit up to 16 columns. This vacuum assist decreases the time spent processing the samples by eliminating gravity drains. Sixteen samples can simultaneously be processed from cleared lysate to elution in under 15 min. Utilizing the inducbile origin of replication, oriV, to increase copy number (Epicentre Technologies), there is generally more than enough DNA for bidirectional sequencing as well as molecular analysis. For one particular fosmid project, yields average about 2.0 ug (~1-3 ug) per 5 mL induced culture, and consistent quality sequence reads of greater than 500 bases have been obtained. Over 90 clones were purified and bidirectionally sequenced producing 156 sequences (out of a potential 180, an 88% sucess rate) in the sequencing project.