Method: Recovery and Purification of Cosmid DNA Using a CsCl2 Gradient

April 26 1990

Matthew S. Holt

Preface:

Large-scale cosmid preps are in essence identical to large-scale plasmid preps with the following exceptions:

1. Inoculate 15 ml LBM+ antibiotic with one colony and incubate at 37 degrees C in a shaker incubator for two hours. Use 2 X Amp in the media if selection is for ampicillin i. e. 200 ug ampicillin per ml media from an Amp stock of 25 mg/ml. Then divide this culture among 4 to 6 flasks containing 350 ml of pre-warmed LBM+ antibiotic (use 2 X Amp) and grow at 37 degrees C for 16-20 hours.
2. Cosmid yields are much smaller than plasmids. One culture is seldom enough to visualize DNA bands even with UV illumination. Better results are achieved by pooling the bands of 4-6 cultures at Day 4 step 1.

3. It often takes 3 or 4 CsCl2 spins to separate the E. coli chromosomal DNA from the cosmid DNA.

4. Vortexing will shear cosmid DNA. After pulling the cosmid band always remove the needle before transferring to the next tube to minimize shearing. Be as gentle as possible.