Screening of M13 mp bacteriophage clones for small inserts by primer extension and insert excision

Abstract

The M13 bacteriophage was adapted by Messing for DNA cloning by insertion of sequences containing a β galactosidase gene with internal restriction sites. Cloning into these sites results in loss of β galactosidase activity, allowing for initial screening of plaques for insert DNA but sometimes producing false positives. In our hands, previously described methods for the further screening of phage for short inserts have limitations that are overcome by the method described here. In our method, a commercially available primer is annealed to single-stranded phage DNA at a sequence that lies immediately 5′ to the cloning site within the β galactosidase gene. Klenow fragment and 4 deoxynucleotide triphosphates are then employed to extend the primer, forming double-stranded DNA, which is cleaved at restriction sites flanking the cloning site. Agarose gel electrophoresis with ethidium bromide staining is used to separate phage from insert DNA, thus demonstrating the presence of cloned insert.