Ecology and Distribution of Thaumarchaea in the Deep Hypolimnion of Lake Maggiore

Table 2

Primer sets and conditions used for 16S rRNA and AmoA genes amplification and cloning of ammonia-oxidizing prokaryotes. An initial denaturation (94°C for 5 min) and final elongation (72°C for 20 min) steps were performed to all reactions. ^(a)Touchdown 0.5°C per cycle, from at 68°C to 60°C; ^(b)+1 sec per cycle. Final elongation = 5 minutes. ^(b)New: reverse primer, specifically targeting Thaumarchaeota (thaum922r: 5′-TTG TGG TGC TCC CCC GCC-3′). f: forward; r: reverse primer; ^(c)primers contained 40-nucleotide-long GC-sequence at the 5′ end for Denaturing Gradient Gel Electrophoresis [31].

Target gene/group	Primer pair	Cycles	Denaturation		Annealing		Elongation		Reference
			°C	s	°C	s	°C	s
Archaea; 16S rRNA	arc344f^(c); arc915r	30	94	60	68^(a)	60	72	90	Stahl and Amann (1991) [24]
Thaumarchaea; 16S rRNA	arc21f; thaum922r^(b)	30	94	60	62	60	72	80	DeLong (1992) [32]; This study
AOB 16S rRNA	CTOf-mix^(c); CTOr	35	92	30	57	60	72	45^(b)	Kowalchuk et al. (1997) [33]
AOB amoA	amoA-1f^(c); amoA-2r	35	94	60	60	90	72	90	Rotthauwe et al. (1997) [34]
AOA amoA	Arch-amoAF^(c); Arch-amoAR	30	94	45	53	60	72	60	Francis et al. (2005) [35]