Restriction analysis of recombination substrate plasmids with EcoRI and HindIII restriction enzymes.

<p><b>A)</b> Scheme of the recombination substrate plasmids. EcoRI and HindIII restriction sites were used to clone the insert containing the two <i>oriT</i> copies, which are shown as black rectangles. The vector-encoded <i>cat</i> gene is indicated as a black arrow. <i>Pcat</i>, <i>cat</i> promoter. <b>B)</b> In not recombined substrate plasmids, we expected a band of 2.3 kb for vector pSU19 and a band of 3.2 kb for the insert containing both <i>oriTs</i> (3.0 kb for pCIG1064, due to the smaller size of the cloned F <i>oriT</i>s). The band of the vector is not detected; instead a band of about 3.5 kb is visible. <b>C)</b> The expected bands are seen in pCIG1028 when IS10 was not present (2.3 kb + 3.2 kb bands for non-recombined and 2.3 kb for recombined plasmid, as the band of 0.4 kb corresponding to the unique <i>oriT</i> is not visible). When IS10 is present, the vector band increases up to around 3.5 kb. HL, Hyperladder I (Bioline), with the size of relevant bands indicated in kb on the left. NR, not recombined. R, recombined.