Thetford2012, sample ID 1.12.D
The DNA of the sample 1.12.D was PCR amplified using the degenerate primers, Fol-degen-for 5′-TCNACNAAYCAYAARRAYATYGG-3′ and Fol-degen-rev 5′-TANACYTCNGGRTGNCCRAARAAYCA-3′. The standard Roche A-adaptor and a unique 10 bp MID tag for each sample (within collection) were attached to the forward primer. Each sample was amplified in three independent reactions and pooled. PCRs were performed in 20 μl reaction volumes containing 2 μl of 10× buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 μM each primer, 0.6 U HotStart Taq DNA polymerase (TaKaRa Biosystems, Ohtsu, Japan), and approximately 60 ng of pooled genomic DNA. We used a touchdown thermocycling profile of 95°C for 2 min; 11 cycles of 95°C for 15 s; 51°C for 30 s; 72°C for 3 min, decreasing the annealing temperature by 1 degree every cycle; then 17 cycles of 95°C for 15 s, 41°C for 30 s, 72°C for 3 min, and a final extension of 72°C for 10 min. We used non-proofreading Taq and fewer, longer cycles to reduce chimera production. For pyrosequencing, PCR products were gel purified by using a Qiagen QIAquick PCR purification kit, quantified by using the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen), pooled and A-amplicon-sequenced on a Roche GS FLX at the Kunming Institute of Zoology.