(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The method used for the isolation of large scale cosmid and plasmid DNA is an unpublished modification (16)of an alkaline lysis procedure (17,18)followed by equilibrium μltracentrifugation in cesium chloride-ethidium bromide gradients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 μltracentrifugation overnight.The ethidium bromide stained plasmid or cosmid DNA bands,equilibrated within the cesium chloride density gradient after μltracentrifugation,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 100μg/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.

2.Harvest the cells by centrifugation at 7000 rpm for 20 minutes in 500 ml bottles in the RC5-B using the GS3 rotor.Resuspend the cell pellets in old media and transfer to two bottles,centrifuge as before,and decant the media.The cell pellets can be frozen at -70degC at this point.

3.Resuspend the cell pellets in a total of 70 ml of GET/Lysozyme solution (35 ml for each bottle)by gently teasing the pellet with a spatula and incubate for 10 minutes at room temperature.(Note: Do not vortex the lysate at any time because this may shear the chromosomal DNA ).

4.Add a total of 140 ml of alkaline lysis solution (70 ml for each bottle),gently mix,and incubate for 5 minutes in an ice-water bath.

5.Add 105 ml of 3M NaOAc,pH 4.8 (52.5 ml for each bottle),cap tightly,gently mix by inverting the bottle a few times,and incubate in an ice-water bath for 30-60 minutes.

6.Clear the lysate of precipitated SDS,proteins,membranes,and chromosomal DNA by pouring through a double-layer of cheesecloth.Transfer the lysate into 250 ml centrifuge bottle,centrifuge at 10,000 rpm for 30 minutes at 4deg C in the RC5-B using the GSA rotor.

For cesium chloride-gradient purification:

7a.Pool the cleared supernatants into to a clean beaker,add one-fourth volume of 50% PEG/0.5 M NaCl,swirl to mix,and incubate in an ice-water bath for 1-2 hours.

8a.Collect the PEG-precipitated DNA by centrifugation in 250 ml bottles at 7000 rpm for 20 minutes at 4degC in the RC5-B using the GSA rotor.

9a.Dissolve the pellets in a combined total of 32 ml of 100:10 TE buffer,5 ml of 5 mg/ml ethidium bromide,and 37 g cesium chloride (Var Lac Oid Chemical Co.,Inc.)(final concentration of cesium chloride should be 1 g/ml).