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A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.

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Sequencing in less than four lanes on a gel, using only one fluorescent label for the four bases (without variations in electrophoretic mobilities or problems with spectral overlap) in one tube was introduced in (1). In that process T7 DNA polymerase and buffer containing magnesium ions were used. By adjusting the ddNTPs ratios the sequence is deduced from the peak magnitudes corresponding to the four bases. The resolution was limited by nonuniformity of the peaks and by decrease of overall labelling due to the two-step process used in that sequencing protocol. In the method described in this note, applicable both to fluorescent and radioactive endlabelling, we have used the one-step protocol with manganese ions in one tube as described (2). This protocol results in uniform overall labelling (within 15% up to base 300). Biochemicals, annealing, termination and stop mixes, as well as diluted enzyme were as described in (4). Briefly, 1.25 itg of M13 ssDNA in 5 Il solution were combined with 2 IL fluorescently labelled primer (2 ,tM) and 6 ILI annealing mix. The mixture was denatured at 65°C for A B C 3 minutes and cooled down to 25°C. Then 4U of T7 DNA polymerase in 2 Il were added and mixed. Nucleotide mixes, as described below, were added and strand synthesis proceeded for 10 minutes at 37°C. The reaction was stopped with 20 IL