Quantitative PCR Analysis of ntrC Mutant

The qPCR analysis was carried out using Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent, Switzerland) and an Mx3000P instrument (Agilent, Switzerland). The cDNA was prepared from an independent biological replicate (of both wild-type and ntrC mutant strains) as previously described [42 (link)]. Each PCR reaction was run in triplicate with 3 dilutions of cDNA (15, 7.5 and 3.75 ng) using 15 μl 2x Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix, and 5 μM of individual primers in a total volume of 30 μl. Fold-changes in transcription and the standard deviation of 9 sample dilutions were calculated using the ΔΔ CT method [43 (link)]. The primary σ factor gene rpoD was used as a reference for normalization. All the primers used are listed in S1 Table.

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Liu Y., Lardi M., Pedrioli A., Eberl L, & Pessi G. (2017). NtrC-dependent control of exopolysaccharide synthesis and motility in Burkholderia cenocepacia H111. PLoS ONE, 12(6), e0180362.

Publication 2017

Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix Mx3000P instrument

Biological Cdna Dilutions Fast green Gene Primers Replicate Strains Transcription σ factor

Corresponding Organization : University of Zurich

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Variable analysis

independent variables

Wild-type and ntrC mutant strains

dependent variables

Fold-changes in transcription

control variables

CDNA prepared from independent biological replicates
PCR reactions run in triplicate with 3 dilutions of cDNA (15, 7.5 and 3.75 ng)
Use of 15 μl 2x Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix
Use of 5 μM of individual primers in a total volume of 30 μl
The primary σ factor gene rpoD used as a reference for normalization

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