Figure 1

Experimental steps for high-throughput LE/RE-spacer mutant loxP screen. Two pools of oligonucleotides containing LE and RE mutants were PCR-amplified to create dsDNA. The LE-mutants were subcloned in pUC19. LE-mutants (in plasmid form) were combined with linear RE oligonucleotides in the presence of Cre recombinase overnight to form the expected 1.8 kb recombination products. These products were PCR amplified with the NotI tailed forward and reverse primers, gel-purified, digested with NotI, re-circularized to reform the pUC19 plasmid and then sequenced.