Large scale sequencing projects using rapidly prepared double-stranded plasmid DNA.
We have developed a simple rapid plasmid DNA mini-preparation method which yields DNA of sufficient quality to be used in large scale sequencing projects. The method, which is a modification of the alkaline method of Birnboim and Doly (1979), requires less than two hours. We have eliminated the use of organic extractions, RNase digestion and alkaline denaturation of the DNA for annealing of the primer. The proportion of supercoiled plasmid DNA obtained is close to 100%. Greater than 80% of the clones yield at least 500 bp of sequence information per primer. The sequencing reactions from these double-stranded templates can be done on both strands using the universal and reverse sequence primers with the usual two reactions per primer, one to read close to the primer and one to read far from it. Thus, each clone yields at least 1 kb of sequence information. The preparation of the templates and the sequencing reactions can be done in less than three hours so that the sequencing gel can be run the same day.
Brun
Y V
YV
Départment de biochimie, Faculté des sciences et de génie, Université Laval, Québec, Canada.
Breton
R
R
Lapointe
J
J
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping
D004247:DNA