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A rapid and versatile method to transfer an insert between single-stranded vectors and reverse its orientation.

We report a novel method to transfer an insert from single-stranded Ml3mpl8 (the donor) into single-stranded pUC1 19 (the recipient); the resultant insert orientation is opposite that found in the donor. The method is performed as follows: The single-stranded recipient vector is linearized at a specific site using an oligonucleotide and restriction enzyme. The resultant termini are annealed to sequences that flank the insert contained in the single-stranded donor vector. The annealed molecules are simply transformed into competent E. coli then incubated on solid media that selects for the recipient. In routine molecular biological work, it is often desirable to reverse the orientation of an insert in a plasmid or phage vector or to transfer the insert into a different vector. The traditional method to manipulate an insert involves digesting double-stranded DNA with restriction enzymes at sites that flank the insert followed by ligation into a second vector, transformation into E. coli, then screening to obtain the clone of interest (1). Not only is this method somewhat time consuming, but problems often arise when appropriate restriction sites are not present. Here we report a method, explained in Figure 1, to transfer an insert between two single-stranded vectors. The vectors tested were M13mpl8 as the 'donor' and pUC1 19 as the 'recipient' but other vector combinations should work (see last paragraph). We believe the requirements of the method are: