A small plasmid for recombination-based screening

Abstract

We reported recently the construction of the 4.4-kb RoK-derived pMADI plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with Co1E1 and therefore permits recombination-based screening of 2 libraries that contain Co1E1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the [pi]-encoding pir gene required for R6K replication. To supply [pi] [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans we employed pPR1[Delta]22pir 116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger 2 inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1(1991) 120-123].