title: masD gene sequencing of AMV825 sediment sample (SAMN03407276)
design: The masD/assA gene was amplified using the primer pair 7757f-1,f-2 (TCG GAC GCG TGC AAC GMY CTG A) / 8543r (TCG TCR TTG CCC CAY TTN GG) or primer pair 7766f (TGT AAC GGC ATG ACC ATT GCG CT) /  8543r (TCG TCR TTG CCC CAY TTN GG) {von Netzer, 2013}. Primers were barcoded and extended with a SfiI restriction site at the 5’ end for ligation with the 454-adapters. For each sample, 8 replicate PCRs (20 μl volume) per primer pair were carried out containing, 0.5 μM primer each, 250 μM dNTPs, 0.3 μg/μl BSA, 1 × PCR buffer, 0.25 U Taq polymerase (5Prime, Germany) under the following conditions: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation (96°C, 1 min), annealing (58°C, 1 min), elongation (72°C, 2 min), and a final elongation step (72°C, 10 min). The replicate PCR reactions were pooled, 800 bp-amplicons extracted from an agarose gel (1.5% w/v) and purified using the MiniElute PCR Purification Kit (Qiagen) according to the manufacturer’s recommendations. Massive parallel tag sequencing of the amplicons was carried out on a 454 Life Sciences GS FLX sequencer.
attributes.tag: SFi Adapter
attributes.value: AGAGTC