3730 dna analyzer

Genomic DNA was isolated by isopropanol precipitation of keratinocyte lysates (lysis buffer was Tris [pH 8] 100 mM, EDTA 5 mM, SDS 0.2%, NaCl 200 mM, and 1 mg/mL proteinase K [Roche Diagnostics, Mannheim, Germany]) and resuspended in Tris/EDTA (TE) buffer. Approximately 20–50 ng genomic DNA was used for PCR amplification. PCR fragments spanning the nuclease target sites were generated with primers F1/R (F1, 5′-gtgagtggtggctgaagcac-3′; and R, 5′-accccaccaaggaaactga-3′). PCR program TD 68-63 was as follows: 94°C for 5 min; 5 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 30 s, decreasing annealing temperature 1°C every cycle; followed by 30 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s; then 72°C for 7 min. PCR products were analyzed in 1.5% agarose gel. Molecular weight marker was IX (Sigma-Aldrich). For sequencing, PCR products were treated with Illustra ExoProStar (GE Healthcare, UK), sequenced using Big Dye Terminator version (v.)1.1 Cycle Sequencing kit (Thermo Fisher Scientific, Waltham, MA) and examined on a 3730 DNA Analyzer (Life Technologies, Carlsbad, CA). Chromatograms were analyzed using Sequencher (Gene Codes, Ann Harbor, MI) and Chromas (Technelysium, Australia) software. Bio-Rad Image Lab Software 6.0 was used for PCR band densitometry.

Genomic DNA isolation and PCR amplification with primers F1/R were performed as described above. PCR products were treated with illustra ExoProStar (GE Healthcare, UK), sequenced using Big Dye Terminator V.1.1 Cycle Sequencing kit (Thermo Fisher, Waltham, MA), and examined on a 3730 DNA Analyzer (Life Technologies, Carlsbad, CA). Chromatograms were analyzed using Sequencher (Gene Codes, Ann Harbor, MI) and Chromas (Technelysium, Australia) software.