Applied biosystems 3500 genetic analyser

We purified the endpoint RT-PCR products with Sephadex^® G-100 (Sigma-Aldrich, St. Louis, MO, USA) columns by centrifugation at 770×g for 3 min and then prepared the sequencing reactions with the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing reactions had a final volume of 10 μl containing 1 μl BigDye^® Terminator v3.1 Ready Reaction Mix, 1.5 μl 5× Sequencing Buffer, 0.2 μM of either the forward (FS778) or the reverse (cFD2) primer and 3 to 10 ng cDNA. The temperature profiles were as follows: 1 min at 96 °C, followed by 25 cycles of 10 s at 96 °C, 5 s at 50 °C and 4 min at 60 °C. We then purified the sequencing products with Sephadex^® G-50 (Sigma-Aldrich) columns by centrifugation at 770×g for 3 min and mixing with 5 μl of HiDi Formamide (Applied Biosystems) and sequenced the prepared cDNA in both directions on an Applied Biosystems 3500 Genetic Analyser (Applied Biosystems). We examined the sequences with the MEGA6 Software [37 (link)] and compared them against the BLAST Nucleotide database [38 (link), 39 ].

PCR products (2.5 μL) were purified using 1 μL ExoSAP-IT enzyme mix (Affymetrix, USA), (incubation at 37°C for 15 min; enzyme deactivation at 80°C for 15 min) and subjected to direct nucleotide sequencing using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, USA) following the manufacturer's instructions. The sequencing was performed on Applied Biosystems 3500 Genetic Analyser (Applied Biosystems, USA). Sequences were analyzed with the Sequencing Analysis Software v5.2.0 (Applied Biosystems, USA) and examined using the Nucleotide BLAST program.
All identified mutations/variants were validated in additional independent round of PCR and once again sequenced.