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 50oC.
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
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