Overnight cultured strains were inoculated in 25 mL M9 medium in a 1% inoculum. After incubation to logarithmic growth phase at 30°C and 150 rpm, the cells were collected for mRNA extraction and processed according to the manual with an RNA pure Bacteria Kit (CWbiotech, Beijing, China). Genomic DNA was then removed for reverse transcription and synthesis of cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWbiotech). Real-time quantitative polymerase chain reaction(RT-qPCR) was performed using ultra SYBR mixtures (CWBiotech) on a CFX96 touch RT-qPCR detection system (Bio-Rad, Hercules, CA, USA). 16S rRNA was amplified using RT-16S-F/RT-16S-R primers and used as an internal reference. The primer pairs of RT-sadA-F/RT-sadA-R, RT-sadB-F/RT-sadB-R, RT-sadC-F/RT-sadC-R, RT-ssuE-F/RT-ssuE-R, RT-rutF-F/RT-rutF-R, RT-cogA-F/RT-cogA-R, RT-sutR-F/RT-sutR-R, and RT-psuK-F/RT-psuK-R were used to amplify sadA, sadB, sadC, ssuE, rutF, cogA, sutR, and psuK, respectively. Relative transcript levels of these genes were calculated using the 2−∆∆CT method (31 (link)), with P. denitrificans DYTN-1 as the control.
Ultra sybr mixtures
Manufactured by CWBIO
Sourced in United States
Ultra SYBR mixtures are a ready-to-use solution for real-time PCR amplification and detection. The mixture contains SYBR Green I dye, a DNA-binding fluorescent dye, along with necessary PCR components, including DNA polymerase, dNTPs, and buffers.
Automatically generated - may contain errors
2 protocols using ultra sybr mixtures
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Quantitative gene expression analysis
2
Quantitative gene expression analysis
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Overnight cultured strains were inoculated in 25 mL M9 medium in a 1% inoculum. After incubation to logarithmic growth phase at 30°C and 150 rpm, the cells were collected for mRNA extraction and processed according to the manual with an RNA pure Bacteria Kit (CWbiotech, Beijing, China). Genomic DNA was then removed for reverse transcription and synthesis of cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWbiotech). Real-time quantitative polymerase chain reaction(RT-qPCR) was performed using ultra SYBR mixtures (CWBiotech) on a CFX96 touch RT-qPCR detection system (Bio-Rad, Hercules, CA, USA). 16S rRNA was amplified using RT-16S-F/RT-16S-R primers and used as an internal reference. The primer pairs of RT-sadA-F/RT-sadA-R, RT-sadB-F/RT-sadB-R, RT-sadC-F/RT-sadC-R, RT-ssuE-F/RT-ssuE-R, RT-rutF-F/RT-rutF-R, RT-cogA-F/RT-cogA-R, RT-sutR-F/RT-sutR-R, and RT-psuK-F/RT-psuK-R were used to amplify sadA, sadB, sadC, ssuE, rutF, cogA, sutR, and psuK, respectively. Relative transcript levels of these genes were calculated using the 2−∆∆CT method (31 (link)), with P. denitrificans DYTN-1 as the control.
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