Preparation of BAC (Bacterial Artificial Chromosome) DNA with CONCERT ™ High Purity Plasmid Purification System1 Courtesy Lisha Xu and Alice C. Young, Molecular and Cell Biology Research and Development, Life Technolgies, Inc., Rockville, MD 20849.

Before Beginning:

Prepare a 20 h culture of BAC containing bacteria in 2X YT and appropriate antibiotic. The OD600 of the final culture should be 5.0 -0.5.

Add RNAse A to E1 to a final concentration of 400 µg/mL.

Increase NaCl concentration in Wash Buffer (E5) from 0.8 M to 0.9 M NaCl by adding 0.58 g NaCl per 100 mL E5. Conductivity should be 72 mS. This increase in salt will reduce the RNA contamination in the BAC prep.

Pre-warm Buffer E6 to 50℃.

NOTE: In this application, we recommend 400 µg RNAse A/mL of Suspension Buffer (E1) because of the extremely low copy number of BACs.

Equilibrate the column with Equilibration Buffer (Buffer E4) Allow the solution.