454 sequencing of Glomeromycota: disturbed and native grassland (Waterton Lakes National Park)
"50 mg ground roots (MoBio Power Plant), NS31 and AML2 (Simon et al. 1992, Lee et al. 2008). 454 sequencing adapters A and B. Adapter A, CCATCTCATCCCTGCGTGTCTCCGACTCAG, was inserted before NS31 and B, CCTATCCCCTGTGTGCCTTGGCAGTCTCAG, before AML2 with 10 nt barcodes. PCR (HotStarTaq Master Mix Kit) 3 ?l template DNA (2 ng/?l), 1 ?l primer (10 ?M), 0.6 ?l bovine serum albumin (20 mg/ml) (S1000 Thermocycler) 95 oC for 15 min, 30X 94 oC 45 sec, annealing 60 oC 45 sec, polymerization 72 oC 1 min & 72 oC 7 min. Purified using QiaQuick, G?nome Qu?bec Innovation Centre purification with Agencourt AMPure XP PCR purification kit, measured and pooled samples in equal concentrations. Sequencing was performed using a half-plate of a Roche GS-FLX Titanium Sequencer."
PC-D-5