Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

A Simple And Efficient Method For Purification Of High-Quality BAC DNA That Is Stable For 2 Years At -20C

Dilara Begum

  EPICENTRE, 726 Post Road, Madison, WI 53711

Purifying enough BAC DNA with sufficient quality and stability for downstream analysis is a significant challenge for many researchers. Limiting factors include the large size and low copy number inherent to this type of clone. We have developed BACMAX™ DNA purification kit for isolating BAC DNA that maximizes both yield and quality without the need for columns, resins, or organic extractions. Moreover, we have varied several parameters to optimize aeration and cell density. The protocol is based on a modified alkaline-lysis procedure that typically yields 0.6 to 25 microgram of BAC DNA from 1.5 to 100 ml of a single-copy BAC culture. Thus, the yield from a 1.5-ml culture is sufficient to perform two end sequences and a fingerprint. This protocol can also be used to isolate at least 15-fold more BAC DNA from cultures of CopyControl BAC clones induced to high-copy number. Successful use of BAC DNA in downstream applications depends not only on the quantity of DNA but the quality. One of the most rigorous tests of BAC DNA quality is whether the DNA remains intact after long term storage. We will demonstrate that BAC DNA isolated with this method and stored for 3 years at -20 degree C showed no significant reduction in quality.