Abi prism 310 genetic analyzer sequencer

Deletion mutation screening was conducted by direct sequencing analysis. PCRs comprised 1 μL of DNA in the presence of 10× Buffer (TaKaRa; Shiga, Japan), dNTP (2.5 mM dNTPmix; TaKaRa), 20 μM forward/reverse primer (AAG deletion R/F; Rikaken; Nagoya, Japan), and rTagDNA polymerase (TaKaRa). The PCR mixture was amplified with PCR Thermal Cycler Dice® Standard (TaKaRa). The conditions of amplification were 94 °C for 1 min; 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and elongation at 74 °C for 1 min; 1 cycle at 72 °C for 7 min. PCR products were purified from the reaction mixture using distilled water. Cycle sequencing was performed using the Big Dye® Terminator v1, Cycle Sequence Kit (Applied Biosystems) up to 20 μL, and then cleaning step and loaded onto an ABI PRISM^® 310 Genetic Analyzer sequencer (Applied Biosystems).