**1.3 Scoring authentic lox-Cre recombinations by unique size of BAC vector DNA upon Not I digestion**

Whether the BAC DNA of reduced size arose from an authentic lox-Cre recombination, as opposed to an internal deletion between sequence repeats or other recombinogenic sites in the genomic insert, is one of the early determinations that need to be made. Note that lox-Cre independent deletions can also be packaged by the P1 headful mechanism when there is no selection for lox site transposition *per se;* such deletions are also efficiently isolated (23). Therefore all our loxP and lox511 transposons have been designed to alter the size of the Not I-Not I BAC-vector DNA band that arises from a Not I enzymatic digest of the deletion clone DNA [see references (23, 24, 27) for discussion]. An end-deletion from Cre-loxP recombination alters the size of BAC vector DNA, whereas deletions arising from within the genomic insert do not. End-deletions made with our lox511 transposons remove the Not I site at the lox511 end in the original BAC, resulting in a linear BAC with no separate vector DNA band (24).

Fig. 3. Legend: FIGE display of an array of BAC deletion clone DNAs. DNA was isolated by the miniprep procedure from clones of a library of end-deleted BACs generated using a loxP transposon. The DNA was digested with Not I enzyme before electrophoresis. Lane 1 shows

**1.3 Scoring authentic lox-Cre recombinations by unique size of BAC vector DNA upon** 

Whether the BAC DNA of reduced size arose from an authentic lox-Cre recombination, as opposed to an internal deletion between sequence repeats or other recombinogenic sites in the genomic insert, is one of the early determinations that need to be made. Note that lox-Cre independent deletions can also be packaged by the P1 headful mechanism when there is no selection for lox site transposition *per se;* such deletions are also efficiently isolated (23). Therefore all our loxP and lox511 transposons have been designed to alter the size of the Not I-Not I BAC-vector DNA band that arises from a Not I enzymatic digest of the deletion clone DNA [see references (23, 24, 27) for discussion]. An end-deletion from Cre-loxP recombination alters the size of BAC vector DNA, whereas deletions arising from within the genomic insert do not. End-deletions made with our lox511 transposons remove the Not I site at the lox511 end in the original BAC, resulting in a linear BAC with no separate vector

a 5 kb ladder, and lane 2 shows the DNA from the starting BAC clone.

**Not I digestion** 

DNA band (24).

Once authenticity of the retrofitted/ end-deleted BAC clone is established, the exact location of insertion of the transposon on genomic DNA is determined by sequencing the BAC DNA directly with unique primer sites located on either the loxP or lox511 transposon ends that remain after deletion formation (13, 24).
