Qiasymphony sp as instrument

Genomic DNA from cell lines and primary fibroblasts was extracted using QIAsymphony SP/AS instruments (QIAGEN) according to the manufacturer’s protocol and quantified on a NanoDROP 2000 spectrophotometer (Thermo Scientific). Polymerase chain reactions (PCRs) were carried out in 25 μl reaction mixtures containing 50 ng of genomic DNA, 1× PCR buffer (Tris–HCl, (NH₄)2SO₄, 15 mM MgCl₂; pH 8.7), 200 μM dNTPs (Thermo Fisher Scientific, R0181) and 0.5 U Taq DNA Polimerase Recombinant (Thermo Fisher Scientific, EP0405) and the following primers (10 pmol each): FOXO3 rs2802292g/t Fw and FOXO3 rs2802292g/t Rv. PCR amplification cycles were carried out at 95°C for 15 min followed by 29 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min, and by a final extension at 72°C for 10 min on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). A total of 5 μl of the amplified products were loaded onto 2% Agarose Standard Low EEO (Sigma, A9539) in 0.5× Tris-Borate-EDTA (TBE) (Serva, 39320.01) and visualized using GelRed™ (Biotium, 41003). Sequencing products were purified by use of the DyeEx™ 2.0 Spin Kit (QIAGEN, 63206) and sequenced on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Primer sequences are listed in ‘Additional Material’.