A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.
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.
D001322:Autoanalysis; D001483:Base Sequence; D003090:Coliphages / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification; D008722:Methods; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004279:DNA, Viral