For all samples, the product of the expected size (1.5 kb) was excised
from the gel (total of 100 mg gel material). The gel fragment was melted
by heating for 5 min at 65°C, and 1 μL was used as template in a second
PCR using the CTO primers (CTOf189-GC, CTO654r), previously
described to amplify specifically a 465-bp fragment of the 16S rRNA
gene from L-Proteobacteria ammonia-oxidising bacteria [17] with the
addition of a 5P GC-clamp