Lambda maxi kit

The RGC-32 genomic locus was isolated as a recombinant bacteriophage λ clone from a 129/Sv mouse genomic library using a mouse RGC-32 cDNA probe (Badea et al., 2002 (link)). The phage isolates obtained after tertiary screening were then purified using a Lambda Maxi kit from Qiagen (Valencia, CA). The lambda DNA obtained was digested with restriction enzymes (NotI, EcoRI, and SalI). The enzyme digests were subcloned into pBluescript SK (Stratagene), and the positive fragments from Southern analysis were sequenced at the University of Maryland’s Biopolymer Lab. A contig was generated on the basis of sequence identity with the mouse genomic sequence published on the NCBI website. Two of the clones resulting from library screening covered exons 3, 4, and 5 and the intron preceding exon 3, and the other two isolated clones covered the rest of the sequence, including exons 1 and 2. The targeting vector consists of: a) the 5′ homology arm of the targeting vector containing a 4-kb Kpn I to Eco RI fragment, ending about 800 bp upstream of exon 1; b) a Frt-PGKNeo-Frt positive selection cassette; c) the 3′ homology arm, spanning about 3 kb, beginning about 300 bp downstream of exon 2; d) the negative selection MC1-TK cassette, and the pBSKS vector backbone. Thus, after homologous recombination, a region spanning exons 1 and 2 is replaced by the FRT-PGKNeo-FRT cassette (Fig. 1A).