Archaeal and bacterial amoA gene abundances were determined by real-time PCR in a RealPlex2 Mastercycler (Eppendorf, Stevenage, UK) using QuantiFast® SYBR® Green PCR Master Mix (Qiagen, Crawley, UK), as described by Thion and Prosser47 (link). Briefly, 20 µl final volume reaction comprised of 0.2 mg ml−1 bovine serum albumin (BSA), 1.5 µM of both primers, 10 µl of QuantiFastTM qPCR master mix (Qiagen, Crawley, UK) and 2 µl DNA template, diluted to reach 5–10 ng µl−1. CrenamoA23f/CrenamoA616r48 (link) and amoA-1F/amoA-2R49 (link) primers were used to amplify AOA and AOB amoA genes, respectively (Supplementary Table 3 ). Two complete standard dilution series, prepared as described by Thion and Prosser47 (link), from 101 to 107 AOA or AOB amoA gene copies, were run for every real-time PCR reaction. Amplification efficiency ranged from 83% to 94% and from 92% to 101% for AOB and AOA, respectively, and only reactions with r2 values ≥0.98 were considered.
Quantifast qpcr master mix
Manufactured by Qiagen
Sourced in United Kingdom, Germany
QuantiFastTM qPCR master mix is a ready-to-use solution for quantitative PCR (qPCR) applications. It is designed to provide fast and reliable detection and quantification of target DNA sequences.
Automatically generated - may contain errors
2 protocols using quantifast qpcr master mix
1
Quantifying Archaeal and Bacterial Ammonia Oxidizers
2
Soil Ammonia Oxidizer Quantification
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DNA was extracted from 0.5 g of soil according to Griffiths et al. (2000) , with modifications (Nicol et al., 2005) , and DNA concentration and purity were measured using a
Nanodrop ND-2000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Abundances of AOA and AOB in soil microcosms were estimated by quantitative PCR (qPCR) amplification of amoA genes using the primers CrenamoA23F/CrenamoA616R (Tourna et al., 2008) and amoA-1F/amoA-2R (Rotthauwe et al., 1997), respectively, with 5 µl of 2 ng µl -1 DNA template in 20 µl final volume reactions. AOA and AOB amoA standards were prepared using Ca. N. sinensis and N. multiformis, respectively, as described in Thion and Prosser (2014) . qPCR assays were performed on a Master cycler® realplex 2 thermocycler (Eppendorf, Germany) with QuantiFast TM qPCR master mix (Qiagen, Crawley, UK) as described in Thion and Prosser (2014) . Amplicon size was verified on a 1% agarose gel electrophoresis and qPCR efficiencies for amplification of AOA and AOB amoA genes were 88 -95% and 90 -92%, respectively, with r 2 values >0.99.
Nanodrop ND-2000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Abundances of AOA and AOB in soil microcosms were estimated by quantitative PCR (qPCR) amplification of amoA genes using the primers CrenamoA23F/CrenamoA616R (Tourna et al., 2008) and amoA-1F/amoA-2R (Rotthauwe et al., 1997), respectively, with 5 µl of 2 ng µl -1 DNA template in 20 µl final volume reactions. AOA and AOB amoA standards were prepared using Ca. N. sinensis and N. multiformis, respectively, as described in Thion and Prosser (2014) . qPCR assays were performed on a Master cycler® realplex 2 thermocycler (Eppendorf, Germany) with QuantiFast TM qPCR master mix (Qiagen, Crawley, UK) as described in Thion and Prosser (2014) . Amplicon size was verified on a 1% agarose gel electrophoresis and qPCR efficiencies for amplification of AOA and AOB amoA genes were 88 -95% and 90 -92%, respectively, with r 2 values >0.99.
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