NosZ primers From -> Henry et al 2006 nosZ1F nosZ1R nosZ2F nosZ2r the pcr conditions from Nakano et al 2008 are for a different nos primer but may be worth trying with the nos primers above: Pure cultures




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nosZ primers
From -> Henry et al 2006
nosZ1F

nosZ1R


nosZ2F

nosZ2R
The PCR conditions from Nakano et al 2008 are for a different nos primer but may be worth trying with the nos primers above:





Pure cultures: Pseudomonas fluorescens C7R12

norB primers
From -> Braker and Tiedje 2003
TABLE 1. Selected primers used for amplification of norB genes

Primera Positionb (nt) Primer sequencec (5_–3_)

qnorB2F 1204–1220 GGN CAY CAR GGN TAY GA

qnorB5R 1466–1444 ACC CAN AGR TGN ACN ACC CAC CA

qnorB7R 1841–1822 GGN GGR TTD ATC ADG AAN CC

cnorB1F 364–380 GAR TTY CTN GAR CAR CC

cnorB2F 553–571 GAC AAG NNN TAC TGG TGG T

cnorB6R 942–925 GAA NCC CCA NAC NCC NGC

cnorB7R 1007–991 TGN CCR TGN GCN GCN GT

a Primers are named by qnorB-targeting genes for quinol-oxidizing nitric oxide

reductase and by cnorB-targeting genes for cytochrome c-oxidizing nitric oxide

reductase; forward and reverse primers are indicated by F and R as the last letter,

respectively.



b Positions correspond to the qnorB gene of Ralstonia eutropha H16 (AF002661)

and the cnorB gene of Paracoccus denitrificans Pd1222 (U28078). nt, nucleotide.



c N _ A, C, G, or T; Y _ C or T; R _ A or G; D _ G, A, or T.

Two combinations, qnorB2F-5R and qnorB2F-7R, yielded the

expected amplification products (262 and 637 bp) for qnorB

from the nondenitrifying Synechocystis sp. strain PCC6803

and denitrifying Ralstonia eutropha H16 and additionally

detected the qnorB type from three denitrifying Alcaligenes

strains.

All other denitrifying strains representing different genera

within the Proteobacteria were targeted by primer combinations

cnorB2F-6R (389 bp) and cnorB2F-7R (454 bp) specific

for cnorB.

PCR conditions from Nakano et al 2008:


Pure cultures:

See Table 2 in Braker and Tiedje, there’s a pretty substantial list of possible cultures

nirK primers
From -> Henry et al 2004
Two degenerated primers (5V–3V) nirK876 (ATY GGC GGV CAY GGC GA) and nirK1040 (GCC TCG ATC AGR TTR TGG TT) were designed to amplify a 165-bp fragment (nirK from Sinorhizoboium meliloti 1021 was used as reference sequence for numbering).
The PCR conditions from Nakano et al 2008 are for a different nirK primer but may be worth trying with the nirK primers above:


Pure Cultures:

Table 2


Bacterial strains used in this study and test of the nirK primer sets to

amplify the copper nitrite reductase

Strains Nir type Result of PCRa

Alcaligenes faecalis ATCC8750 Cu (2,0) +

Achromobacter cycloclastes ATCC21921 Cu (0,0) +

Bradyrhizobium japonicum 526 Cu (0,2) +

Rhizobium meliloti Cu (1,0) +

Rhodobacter sphaeroides DSM158 Cu (2,0) +

Pseudomonas fluorescens C7R12 cd1 0

Numbers of mismatches of the nirK sequences from reference

strains with forward and reverse primers are indicated in

parenthesis.

a +, visible band of the expected size; _, no visible band; 0,

non-specific bands.




nirS primers
From Throbeck -> 2004
cd3AF

R3cd


PCR conditions from Nakano et al 2008:


Pure culture: Any of these would work according to Table 1.
Alcaligenes denitrificans CCUG 407T ) ) ) ) ) ) ) ) ) ) ) + ) ) ) +

Comamonas testosteroni CCUG 1426T ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) +

Paracoccus denitrificans CCUG 13798 + + + + + ) ) + + M + + + + + +

Paracoccus denitrificans CCUG 30144 ) + ) ) ) ) ) ) ) ) ) ) + + + +

Paracoccus denitrificans Pd1222a + + + + ) ) + ) ) ) + ) ) + + +

Pseudomonas aeruginosa CCUG 241 + + + + + + + + + M ) + + + + +

Pseudomonas aeruginosa Mi11b + + + + + + + + + ) + + + + + +

Pseudomonas fluorescens ATCC 33512 + ) ) ) + ) ) ) ) M + + ) ) + +

Pseudomonas fluorescens Mi32b + + + + + + ) + + ) ) ) + + + +

Pseudomonas stutzeri ATCC 14405 + + + + + + + M + M + + + + + +

Pseudomonas stutzeri CCUG 29240 + + + + + + + + + ) + + + + + +

Ralstonia eutropha ATCC 17699 ) + + ) ) ) ) + + ) ) ) ) ) ) +



Ralstonia eutropha CCUG 13724 + + + + + + M M ) ) + + + ) + +


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