Quantitect sybr green qpcr mix

Total RNA was extracted using the QIAzol Lysis reagent (Qiagen) following the manufacturer’s instructions. Prior to cDNA synthesis, samples were treated with DNase (Thermo Fisher Scientific). Reverse transcription was performed using SuperScript II (Thermo Fisher Scientific) reverse transcriptase and Oligo(dT)12-18. Real-time PCR was performed using a Mastercycler ep realplex (Eppendorf) and QuantiTect SYBR Green qPCR mix (Qiagen) using the primers listed in S3 Table. Relative transcript levels were calculated according to Vandesompele and colleagues (2002) [37 ].

Within 24h of collection, water samples (1 L) were filtered (0.45 micron filters, Sartorius) and frozen until processed. DNA was extracted from filters and swabs using the FastDNA soil kit (MPBio, Australia) following the manufacturers’ instructions. Bacterial load was measured using a SYBR-based qPCR assay targeting the 16s rRNA gene with PCR primers 331-f and 797-r [30 (link)] and using the QuantiTect SYBR Green qPCR mix (Qiagen, Australia) resulting in a qPCR efficiency of 90%. The delta Ct method was used for relative quantification and a positive control was included in each run for inter-run comparisons. Five DNA extraction negative controls on filters (#3) and swabs (#2) with no water or biofilm added were also processed. The DNA was sent to the Australian Centre for Ecogenomics (ACE, https://ecogenomic.org/) for 16s rRNA gene amplicon sequencing.