Table 1. Primers and PCR conditions used in quantitative PCR† and DGGE# with GC clamp ggcggcgcgccgcccgccccgcccccgtcgccc added to the 5‘ end*.

gene / primer / sequence / product (bp) / qPCR primer (nM) / reference
16S / 331F#† / *tcctacgggagg cag cag t / 194 / 500 / 1
534r#† / attaccgcggctgctgg / 500 / 2
nirK / nirK876† / atyggcggv cay ggcga / 165 / 5000 / 3
nirK1040† / gcctcgatcagrttrtggtt / 5000
nirKFIaCu# / atcatggtsctgccg cg / 472 / 4
nirKR3Cu# / *gcctcgatcagrttgtggtt
nirS / nirS_cd3a-F#† / gtsaacgtsaag gar acsgg / 410 / 2500 / 5
nirS_R3cd-R#† / *gas ttcggrtgsgtcttg a / 2500 / 6
nosZ / nosZ2R† / cakrtgcaksgcrtggcagaa / 268 / 5000 / 7
nosZ2F† / cgcracggcaasaaggtsmssgt / 2500
nosZ1622RC# / *cgsaccttsttg ccs tyg cg / 453 / 6
nosZF# / cgytgttcmtcgacagccag / 8

To reduce heterogeneity between individual DNA extracts, each DNA sample was made from two separate aliquots of 250 mg soil from each sieved core sample used, and then pooled.

For DGGE profiling duplicate PCR reactions were carried out for each of the three replicate samples. PCR amplifications for DGGE were performed in 20µl volumes with 200 µM dNTPs, 2.5 mM MgSO4, 0.2 µM Primers for 16S rRNA, nirK and nosZand 0.4 µM for nirS primers, 3% DMSO for nosZ and 0.5µg BSA for nirS and nirK. The 16S rRNA, nirK,nirS and nosZ products were run at 60 °C on 8% acrylamide gels with 30-60%, 30-60%, 30-45% and 30-50% denaturing conditions, respectively. DGGE PCR cycling conditions - initial denaturing step 98oC (45sec) followed by 30 cycles: annealing temperature 64oC (30 sec);extension temperature 72oC (30 sec); denaturing 98 oC (45sec). A final extension step at 72oC (10 min) was included.

For qPCR analysis triplicate PCR reactions were carried out for each of the three replicate samples. qPCR cycling conditions - initial activation for HotStarTaq DNA polymerase at 95oC (15 min) followed by 40 cycles: annealing temperature 58oC (18 sec); extension temperature 72oC (42 sec); denaturing 95 oC (15sec). A final dissociation curve was generated to determine the quality of the applified products.

References

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