Total RNA from E. coli NC101 grown in LB for 7 h was isolated, and contaminating DNA was removed using DNase I (Biobasic, Ontario, Canada) for 30 min at 37 °C, followed by RNA extraction using the Total RNA Mini-Preps Kit (Biobasic). Reverse transcription PCR was performed on cDNA reverse transcribed from 50 ng RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Real time PCR was performed using the enzyme PowerUp™ SYBR™ Green Master Mix (Thermo Fisher) using the RG 3000A R (Qiagen, Québec, Canada). Primers used are presented in Additional Table 1 . Relative quantitation was performed using standard curves constructed from serial dilutions of PCR products [22 ]. mRNA expression for each gene was determined by direct comparison with the standard curve of the specific target generated in each PCR run. Expression levels of clbA, clbB, clbQ and clbR were normalized to 16S rRNA.
Total rna mini preps kit
Manufactured by Bio Basic
Sourced in Canada
The Total RNA Mini-Preps Kit is a laboratory equipment designed for the rapid and efficient extraction of total RNA from a variety of sample types. It employs a silica-based membrane technology to capture and purify RNA molecules, providing a reliable and consistent method for RNA isolation.
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2 protocols using total rna mini preps kit
1
qPCR Analysis of Enterobacterial Cluster Genes
2
Quantifying clbA Expression in E. coli
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Total RNA from E. coli NC101 grown in LB for 7 h was isolated, and contaminating DNA was removed using DNase I (Biobasic, Ontario, Canada) for 30 min at 37 °C, followed by RNA extraction using the Total RNA Mini-Preps Kit (Biobasic). Reverse transcription PCR was performed on cDNA reverse transcribed from 50 ng RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). For determination of the clbA transcript levels the primer pair RT-4711up/RT-4711lp was used (7) ; and as an internal standard the transcript level of the 16S rRNA gene was amplified with primer pair 16s1114F/16s1275R (Supplementary Table 1). Real time PCR was performed using the enzyme PowerUp™ SYBR™ Green Master Mix (Thermo Fisher) using the RG 3000A R (Qiagen, Quebec, Canada).
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