PAGE 5 OF 9 from a plate were pooled and processed for making end- deletions as described earlier for wild-type loxP transposon- transformed BACs (28,34). BAC deletions were selected on plates that contained chloramphenicol only. Miniprep DNA isolated from deletion clones were digested with NotI enzyme before analysis by FIGE (29). Lanes 3–15 of Figure 2 shows DNA isolated from clones deleted from the loxP511 end of insert in BAC RP23-444K15. Lane 1 displays an intra-insert deletion independent of loxP-Cre (28), with the vector DNA fragment size identical (8.8 kb) to starting BAC (lane 17). Deletions from the loxP511 end were also made with pTnLoxP511(B)RSVneo 2 (Figure 1) in a subset of the BAC clones. Lanes 19–30 Figure 2, shows NotI digested DNA from a set of these deletions with the BAC vector frag- ment now at 10.5 kb. Specificity of loxP-Cre recombinations during deletion formation: using size of pBACe3.6 vector DNA fragment with NotI as a diagnostic The two Not I sites in pBACe3.6 vector are located asymmet- rically with respect to the loxP sites: the NotI sites at positions 2,849 and 11 583 are 1.978 and 0.142 kb in front of the wild- type and mutant loxP511 sites, respectively (35) (Figure 3). Transposons pTnLoxP511(B)RSVneo 2 and pTn(RSVneo 2)/ loxP would substitute the BAC vector NotI site with one from the transposon after recombining the respective loxP sites (Figures 1 and 3). Because the NotI sites in both transposons are 1.7 kb away from their respective loxP sites, they would yield BAC vector DNA NotI-fragments of different size in clones deleted from the wild-type or the mutant loxP511 ends of insert: recombinations with either the mutant 511 or wild- type loxP sites in the BAC would produce a 10.5 or 8.4 kb Nucleic Acids Research, 2005, Vol. 33, No. 13 e118 vector DNA fragment with NotI, respectively, (lanes 19–30 and 16–30 in Figures 2 and 4, respectively). Note that frag- ments of 10.5 and 8.4 kb did not appear together during a deletion experiment with either of these transposons, demon- strating the exclusivity of the recombination reactions. It cor- roborates the selectivity observed during the P1 transductions described above. Deletion-end-sequencing with Tn-based primers also support the conclusion. Deletions from the wild-type loxP end of insert DNA with pTnMarkerless2, described earlier (28), yield the expected vector DNA NotI-fragment of 6.8 kb (lanes 1–13, Figure 4). Intra-insert deletions independent of LoxP-Cre recombination are isolated only when using markerless transposons (28), because the probability of isolating one in conjunction with a transposon insertion is low, and can be easily identiﬁed by their BAC vector DNA fragment with NotI: it is the same size as the starting BAC, as shown in lane 1 of Figure 2. Note that markerless transposons cannot be used for second round deletions because the starting clone would be less than P1- headful size (28). A new wild-type loxP transposon pTn(RSVneo 2)/loxP was used to make deletions from the wild-type end of insert DNA, producing the expected 8.4 kb BAC vector DNA fragment with NotI (lanes 16–30, Figure 4). Truncating insert DNA from both ends sequentially in the same BAC Trimming either end of insert DNA in pBACe3.6 derived clones was readily achieved using transposons speciﬁc for the respective ends. Both ends in the same clone can be deleted similarly except that the second round of deletions must neces- sarily be performed with a resistance gene marker-containing Figure 3. Schematic representation of deletions made sequentially from the (i) mutant loxP511 end of genomic DNA insert generated with pTnLoxP511(B)marker- less 1 (top panel) and (ii) wild-type loxP end with pTn(RSVneo 2)/loxP (bottom panel). The transposon is shown as the triangle with the locations of NotI sites indicated. Note that Cre mediated deletions generated with the loxP511 sites would substitute a NotI with a PmeI site (top panel). Deletions using the wild-type loxP sites would retain the NotI site as shown in the bottom panel.