(adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu)
A. Large scale double-stranded DNA isolation
The method used for the isolation of large scale cosmid and plasmid DNA is an unpublished modification (16) of an alkaline lysis procedure (17,18) followed by equilibrium ultracentrifugation in cesium chloride-ethidium bromide gradients (1). Briefly, cells containing the desired plasmid or cosmid are harvested by centrifugation, incubated in a lysozyme buffer, and treated with alkaline detergent. Detergent solubilized proteins and membranes are precipitated with sodium acetate, and the lysate is cleared first by filtration of precipitate through cheesecloth and then by centrifugation. The DNA-containing supernatant is transferred to a new tube, and the plasmid or cosmid DNA is precipitated by the addition of polyethylene glycol and collected by centrifugation.
The DNA pellet is resuspended in a buffer containing cesium chloride and ethidium bromide, which is loaded into polyallomer tubes and subjected to ultracentrifugation overnight. The ethidium bromide stained plasmid or cosmid DNA bands, equilibrated within the cesium chloride density gradient after ultracentrifugation, are visualized under long wave UV light and the lower band is removed with a 5 cc syringe. The intercalating ethidium bromide is separated from the DNA by loading the solution onto an equilibrated ion exchange column. The A260 containing fractions are pooled, diluted, and ethanol precipitated, and the final DNA pellet is resuspended in buffer and assayed by restriction digestion as detected on agarose gel electrophoresis.
During the course of this work several modifications to the above protocol were made. For example, initially cell growth times included three successive overnight incubations, beginning with the initial inoculation of 3 ml of antibiotic containing media with the plasmid or cosmid-containing bacterial colony, and then increasing the culture volume to 50 ml, and then to 4 l. However, it was observed that recombinant cosmid DNA isolated from cell cultures grown under these conditions, in contrast to recombinant plasmid DNA, was contaminated with deleted cosmid DNA molecules. However, these deletions are avoided by performing each of the three successive incubations for eight hours instead of overnight, although a slight yield loss accompanied the reduced growth times.
Recently, a diatomaceous earth-based (19-22) method was used to isolate the plasmid or cosmid DNA from a cell lysate. The cell growth, lysis, and cleared lysate steps are performed as described above, but following DNA precipitation by polyethylene glycol, the DNA pellet is resuspended in RNase buffer and treated with RNase A and T1. Nuclease treatment is necessary to remove the RNA by digestion since RNA competes with the DNA for binding to the diatomaceous earth. After RNase treatment, the DNA containing supernatant is bound to diatomaceous earth in a chaotropic buffer of guanidine hydrochloride by incubation at room temperature. The DNA-associated diatomaceous earth then is collected by centrifugation, washed several times with ethanol buffer and acetone, dried, and then resuspended in buffer. The DNA is eluted during incubation at 65degC, and the DNA-containing supernatant is collected after centrifugation and separation of the diatomaceous earth particles. The DNA recovery is measured by taking absorbance readings at 260 nanometers. After concentration by ethanol precipitation, the DNA is assayed by restriction digestion.
Protocol
1. Pick a colony of bacteria harboring the plasmid or cosmid DNA of interest into a 12 X 75 mm Falcon tube containing 2 ml of LB media supplemented with the appropriate antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37deg C 8-10 hours with shaking at 250 rpm. Transfer the culture to an Ehrlenmeyer flask containing 50 ml of similar media, and incubate further for 8-10 hours. Transfer 12.5 ml of the culture to each of 4 liters of similar media, and incubate for an additional 8-10 hours.
