A comparative study of naphthalene biodegradation in Temperate and Arctic marine microcosms, based on degradation rate measurements and microbial community composition analysis
Partial 16S rRNA gene was amplified using universal forward, 341f  (5’-CCTACGGGAGGCAGCAG-3’) and reverse, 907r (5’-CCCCGTCAATTCCTTTGAGTT-3’) primers. The fusion primers were designed according to Roche 454 guidelines for unidirectional sequencing with Lib-L chemistry. All DNA samples were amplified in 50 µL PCR reactions (4 tubes per sample). Each reaction contained10 µL reaction buffer (BioRad), 1 mM MgCl2, 0.4 mMd’NTPs, 0.5 µM of each primer, 1U of iProof high fidelity DNA polymerase (BioRad), 1 µL of each DNA sample and molecular grade water. PCR condition were: activation step at 98 °C for 2 min, denaturation at 98 °C for 30 sec, annealing at 57 °C for 30 sec and elongation at 72 °C for 1 min. A final elongation at 72 °C for 7 min was performed. PCR cycle numbers (26-30) were optimized previously to give an approximately similar yield for each sample.All reaction products from the same sample were then pooled and concentrated to a volume of 50 µL using MinElute kit (Quiagen). Samples were run on agarose gel and DNA was extracted from the gel with MinElute Gel Extraction kit (Quiagen). All samples were pooled in equimolar amounts.