Preparation of Single-Stranded Phagemid DNA Without Chromosomal DNA Contamination

During the culture of phagemidcontaining bacteria infected with M13KO7 helper phage, many host strains such as JM109 and XL1-Blue release their chromosomal DNA into the medium because of lysis of bacteria. This results in chromosomal DNA contamination of the preparation of single-stranded DNA (ssDNA) (2,5). A certain strain of E. coli, DH11S, has been engineered to overcome this problem (2). However, because strains such as JM 109 and XL-Blue remain widely used, we developed a simple method for preparation of ssDNA without chromosomal DNA contamination from conventional strains. The principles of the method are denaturation and neutralization introduced to treat enriched phagemids as in plasmid DNA preparation (4). With this treatment, ssDNA can be renatured to its original status, while protein and chromosomal DNA remain denatured and in a precipitated form. Therefore, soluble ssDNA can be separated from protein and chromosomal DNA by centrifugation. Since DNA selectively binds to silica (1,3), it can be separated from trace protein contaminants by treating the recovered ssDNA solution with silica followed by washing. A single colony of male E. coli strain containing the phagemid, picked from a fresh selective medium plate, was suspended in 3 mL of LB broth containing an appropriate antibiotic and incubated overnight. One hundred microliters of the overnight culture were added to 5 mL of tryptone buffered glucose (TBG) (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 55 mM K2HPO4 and 20 mM glucose) containing the antibiotic. After incubation with constant shaking at 37°C for 50 min, the bacteria were infected by the addition of 40 μL of M13KO7 helper phage (ca. 1011 plaque-forming units [pfu]/mL) and incubated for an additional 6 h. The culture supernatant was then harvested by centrifuging the cells at 12 000× g at 4°C for 15 min. The supernatant was placed in a clean tube and centrifuged again as above. To precipitate the phagemids, 0.3 mL 2.5 M NaCl in 20% polyethylene glycol (PEG 8000) was added to 1.2 mL of the supernatant in a microcentrifuge tube. The mixture was left on ice for 15 min and centrifuged at 12 000× g at 4°C for 15 min, and the supernatant was discarded. The Wizard plasmid DNA minipreparation kit (Promega, Madison, WI, USA) was used for phagemid suspension, denaturation, neutralization and silica absorption as follows: The phagemid pellet was resuspended by adding 200 μL of the cell resuspension solution (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 100 mg/mL RNase A). Two hundred microliters of the cell lysis solution (0.2 M NaOH, 1% sodium dodecyl sulfate [SDS]) were added to denature the DNA, followed by the addition of 200 μL of the neutralization solution (1.32 M potassium acetate, pH 4.8). The mixture was centrifuged at 15 000× g for 5 min. The supernatant was decanted into a clean microcentrifuge tube, 1 mL of the Wizard minipreparation DNA purification resin was added to absorb the DNA, and the tube was inverted 3–4 times to mix the contents. A 3-mL disposable syringe with the plunger removed was attached to the Luer-lock extension of a minicolumn. The resin/DNA mixture was pipetted into the syringe and the plunger inserted slowly to introduce the slurry into the minicolumn. The syringe was then detached from the minicolumn, and the plunger was removed. The syringe barrel was reattached to the minicolumn and filled with 2 mL of column wash solution, which was pushed through the minicolumn. To dry the resin, the minicolumn was removed and transferred to a microcentrifuge tube and spun at 12 000× g for 2 min. Thirty microliters of TE (10 mM Tris, 1 mM EDTA, pH 7.8) were applied to the minicolumn, which had been transferred to a new microcentrifuge tube. After 1 min, the DNA was eluted by centrifugation at 12 000× g for 1 min. Three microliters of the sample were analyzed by electrophoresis on 1% agarose gel.