Rapid and reliable dideoxy sequencing of double-stranded DNA.
We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D005822:Genetic Vectors; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D013698:Templates, Genetic
D004274:DNA, Recombinant; D010761:Phosphorus Radioisotopes; D004262:DNA Restriction Enzymes