Bacterial strains were cultured and β-galactosidase assay was performed, as described above. Simultaneously, aliquots of the same culture were taken and total RNA was extracted, as described previously71 (link). RNA concentrations were measured spectrophotometrically and 15 μg of RNA was resolved on 6% polyacrylamide gel electrophoresis. RNA was transferred to 0.2 μm pore size Nytran N (Whatman) membrane, as described previously72 . Membrane was prehybridized for 45 min in ULTRAhyb (Ambion) solution at 42 °C. Blots were probed overnight with 5′-biotinylated SgrS-bio or ssrA-bio probes specific for SgrS sRNA and 5 S rRNA, respectively (Supplementary Table 2 ). BrightStar BioDetect kit (Ambion) was used for detection. ImageJ (National Institutes of Health73 ) was used to measure band densities from two independent experiments.
Nytran n
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The Nytran N is a versatile laboratory equipment designed for filtration and separation applications. It is a high-quality nylon membrane that offers consistent performance and reliability for a variety of research and analytical tasks.
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4 protocols using nytran n
1
Quantitative Northern Blot Analysis
2
Bacterial Conjugation: Resistance Traits Transfer
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Based on antibiograms, agarose gel analysis of plasmid preparations, and integron analysis, representative isolates of Shigella were selected to study transferability of their resistance traits to the recipient by conjugation.36 (link),45 (link)
E. coli XL1-BLUE (resistant to tetracycline and NAL) or E. coli J53 KACC 16628 (resistant to sodium azide) was used as a recipient. Briefly, the recipient and donor were mixed in a ratio of 1:1 on a sterile 0.45 µm nylon membrane (Nytran N, Whatman) and incubated overnight for mating on Luria-Bertani agar at 37°C. Transconjugants were selected on MacConkey agar containing appropriate antibiotics and tested for acquired resistance traits by determination of their antibiotic susceptibility profiles.
E. coli XL1-BLUE (resistant to tetracycline and NAL) or E. coli J53 KACC 16628 (resistant to sodium azide) was used as a recipient. Briefly, the recipient and donor were mixed in a ratio of 1:1 on a sterile 0.45 µm nylon membrane (Nytran N, Whatman) and incubated overnight for mating on Luria-Bertani agar at 37°C. Transconjugants were selected on MacConkey agar containing appropriate antibiotics and tested for acquired resistance traits by determination of their antibiotic susceptibility profiles.
3
Northern Blot Analysis of SgrS RNA
4
Quantification of sRNA Expression
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Bacterial strains were cultured and β-galactosidase assay performed as described above.
Simultaneously, aliquots of the same culture were taken and total RNA was extracted as described previously 72 . RNA concentrations were measured spectrophotometrically and 15 µg of RNA were resolved on 6% polyacrylamide gel electrophoresis. RNA was transferred to 0.2 µm pore-size Nytran N (Whatman) membrane as described previously 73 (link) . Membrane was (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 9, 2020. ; https://doi.org/10.1101/2020.07.24.215095 doi: bioRxiv preprint prehybridized for 45 min in ULTRAhyb (Ambion) solution at 42°C. Blots were probed overnight with 5'-biotinylated SgrS-bio or ssrA-bio probes specific for SgrS sRNA and 5S rRNA respectively (Supplementary Table 2). BrightStar BioDetect kit (Ambion) was used for detection. ImageJ (National Institutes of Health; 74 (link) ) was used to measure band densities from four independent experiments.
Simultaneously, aliquots of the same culture were taken and total RNA was extracted as described previously 72 . RNA concentrations were measured spectrophotometrically and 15 µg of RNA were resolved on 6% polyacrylamide gel electrophoresis. RNA was transferred to 0.2 µm pore-size Nytran N (Whatman) membrane as described previously 73 (link) . Membrane was (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 9, 2020. ; https://doi.org/10.1101/2020.07.24.215095 doi: bioRxiv preprint prehybridized for 45 min in ULTRAhyb (Ambion) solution at 42°C. Blots were probed overnight with 5'-biotinylated SgrS-bio or ssrA-bio probes specific for SgrS sRNA and 5S rRNA respectively (Supplementary Table 2). BrightStar BioDetect kit (Ambion) was used for detection. ImageJ (National Institutes of Health; 74 (link) ) was used to measure band densities from four independent experiments.
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