Amersham hybond nylon membrane

Ten micrograms of genomic DNA from the polymerase chain reaction (PCR)–positive ES clones were digested with restriction enzyme, then separated by 0.8% agarose gel electrophoresis, and transferred to Amersham Hybond nylon membrane (RPN1210B, GE Healthcare), and then Southern blot hybridization experiments were performed using dioxigenin-labeled probes. Visualization was done by exposing to x-ray film for 30 min. PCR primers for generating these probes were as follows: probe 1 (outside short arm): 5′ GCA ATC CGT TTT AGG ACT GCG ATG TAC GAG AC 3′ and 5′ GCT ACA TTC ACA CGA GCA TCC AGG AGG C 3′ to detect an 8.7-kb WT and 11.5-kb mutant Bam HI band; probe 2 (neo cassette): 5′ CGG CAG GAG CAA GGT GAG ATG AC 3′ and 5′ CGC TTG GGT GGA GAG GCT ATT CG 3′; probe 3 (inside long arm): 5′ CAG AGA GCA GCA GAG AAG TGT TAG CTC TTT GGG 3′ and 5′ GTA AGT GTC TGT CCC GCT CGT GGT CA 3′ to detect a 4-kb WT and 7-kb mutant Hind III band.

Ten micrograms of genomic DNA from the polymerase chain reaction (PCR)–positive ES clones were digested with restriction enzyme, then separated by 0.8% agarose gel electrophoresis, and transferred to Amersham Hybond nylon membrane (RPN1210B, GE Healthcare), and then Southern blot hybridization experiments were performed using dioxigenin-labeled probes. Visualization was done by exposing to x-ray film for 30 min. PCR primers for generating these probes were as follows: probe 1 (outside short arm): 5′ GCA ATC CGT TTT AGG ACT GCG ATG TAC GAG AC 3′ and 5′ GCT ACA TTC ACA CGA GCA TCC AGG AGG C 3′ to detect an 8.7-kb WT and 11.5-kb mutant Bam HI band; probe 2 (neo cassette): 5′ CGG CAG GAG CAA GGT GAG ATG AC 3′ and 5′ CGC TTG GGT GGA GAG GCT ATT CG 3′; probe 3 (inside long arm): 5′ CAG AGA GCA GCA GAG AAG TGT TAG CTC TTT GGG 3′ and 5′ GTA AGT GTC TGT CCC GCT CGT GGT CA 3′ to detect a 4-kb WT and 7-kb mutant Hind III band.