A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing.

The alkaline-lysis mini-preparation method of isolating plasmid DNA includes several time-consuming steps. Here we provide a shortened protocol in which the volumes and incubation times have been altered (in particular the ethanol precipitation step) and the phenol extraction and RNAse digestion omitted completely. Recently a mini-prep method has been published requiring the use of caesium chloride and ethidium bromide which then have to be carefully removed. Wong et al. describe SephacrylTM spin columns and state that these may need to be run more than once. Both of these extra procedures add to the expense, and are time-consuming. They are not required in the method we describe here. Our method yields high-quality DNA that can be readily sequenced by the dideoxy chain termination method (Figure 1 shows a portion of DNA sequence from a recombinant pBluescriptll KS plasmid). For practical convenience the method is presented in a simple step-by-step protocol. 1. Inoculate a single 'tooth-picked' bacterial colony into 3 ml of a rich growth medium e.g. Terrific broth (TB), containing antibiotic as appropriate, and incubate with vigorous shaking at 37°C overnight. 2. Decant 2 ml of the overnight culture into an EppendorfTM tube, and spin in a benchtop centrifuge at 13,000 rpm for 1 minute. 3. Completely remove the supernatant from the bacterial pellet, by aspiration. 4. Carefully resuspend the pellet in 200 /tl GTE solution. 5. Add 400 /tl of a freshly prepared solution of 0.2 M NaOH/1 % SDS, and invert several times before placing on ice for 5 minutes. 6. Add 300 /tl of 3 M potassium acetate (pH 4.8), and invert several times before replacing on ice for 5 minutes. Do not vortex. 7. Centrifuge at 13,000 rpm for 5 minutes, and remove supernatant to a clean 2 ml EppendorfTM tube. 8. Add 1 volume (900 /tl) of absolute ethanol to the supernatant, vortex briefly, and immediately centrifuge at 13,000 rpm for 5 minutes. 9. Carefully discard the supernatant, and wash the plasmid pellet with 2 ml of 70% ethanol (optional), before a final centrifugation at 13,000 rpm for 2 minutes. 10. Discard the supernatant, and dry the pellet under reduced pressure. 11. Resuspend the DNA pellet in 40 /tl of sterile TE. The DNA is then ready for sequencing or restriction enzyme digestion. 12. The DNA is alkali-denatured and the contaminating RNA 300 .