PCR amplification of ureC: freshwater bacteria
PCR protocol: Reactions contained 1 uM of the forward primer (HEDWIG/ 5’ GCTATCGGTCTCAAACTTCAYGARGAY- TGGGG-3), 1 uM of the reverse primer (5’-GCAATACCATGCGCAATC- GCNGCNGGRTTDATNGT-3’), template DNA, 0.5 uM of 2X EconoTaq DNA Polymerase (Lucien), and sterile PCR water. PCR was carried out in a cycle of 1 min at 80oC, followed by 24 cycles of: denaturing for 30 s at 95oC, annealing for 1 min at 55oC initially, decreasing 0.3oC per cycle (end at 47.5oC at the last cycle), elongation of 2 min at 72oC. After the 24 cycles, and a 10 min final elongation at 72oC. PCR clean-up using the UltraClean® PCR Clean-Up Kit.