Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Zhang, Shenyuan; Cahalan, Michael D.

doi:10.3791/247

Cited by 20 publications

(13 citation statements)

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“…Plasmid extraction was performed on all ESBL-producing strains using Column-Pure Plasmid Miniprep Kit (Applied Biological Materials (ABM); Canada) [15] . Extracted plasmid DNA was stored at −20 °C.…”

Section: Methodsmentioning

confidence: 99%

Fecal carriage of ESBL-producing <em>Escherichia coli</em> in Egyptian patients admitted to the Medical Research Institute hospital, Alexandria University

et al. 2020

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Commensal ESBL-producing E. coli represent a reservoir for resistance genes therefore, their detection is crucial to restrain the spread of beta-lactam resistance. Hence, the aim of the present study was phenotypic and genotypic characterization of commensal ESBL-producing E. coli obtained from the stool of patients at the time of admission and at the time of discharge from the Medical Research Institute hospital. A total of 70 E. coli isolates were collected from 35 patients and were categorized into Group A (samples obtained on admission) and Group B (samples obtained at the time of discharge). Phenotypically, 30 isolates were ESBL producers (40% of E. coli isolates collected on admission and 45.7% of the strains obtained at the time of discharge were ESBL producers). Most of them harbored one to three plasmids with sizes ranging from one kbp to ten kbp. Upon genotypic investigation, bla CTX-M was the most detected gene in 80% of ESBL strains, followed by bla TEM in 53.3% and the least detected was bla SHV in only 13.3%. By comparing group A and group B, ten patients were found to carry commensal ESBL-producing E. coli , in two patients these isolates carried ESBL genes that were identical on admission and on discharge. However, in eight patients, these isolates carried different ESBL genes, which were newly harbored during hospital stay. The high abundance of MDR commensal E. coli 48.57% together with the presence of 42.86% ESBL-producing commensal E. coli among our isolates represents an alarming threat, as they are frequently associated with the increased risk of infection, higher costs and longer hospital stay.

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Section: Methodsmentioning

confidence: 99%

Fecal carriage of ESBL-producing <em>Escherichia coli</em> in Egyptian patients admitted to the Medical Research Institute hospital, Alexandria University

et al. 2020

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“…Transform the plasmid into DH5α E. coli cells and plate onto LB agar plates with the relevant antibiotic. Select multiple colonies, mini-prep each one, and subject each clone to Sanger sequencing to characterize each deletion allele 28–30 . Repeating the PCR test for deletion after the initial screen ensures that the correct clone was selected and reproducibility of results.…”

Section: Protocolmentioning

confidence: 99%

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Bauer

Canver

Orkin

2015

JoVE

124

100

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SHORT ABSTRACT CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9. LONG ABSTRACT The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

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“…In brief, the genomic DNA was isolated from the culture using the QIAamp DNA purification kit from Qiagen [Zhang and Cahalan, 2007]. The fragment of the 16S rDNA gene was amplified in an Eppendorf thermal cycler from the isolated DNA in the presence of 8F (5′ AGA GTT TGA TCC TGG CTC AG 3′) and 1492R (5′ ACG GCT ACC TTG TTA CGA CTT 3′) primers [Siqueira and Rôças, 2005].…”

Section: S Rdna Analysismentioning

confidence: 99%

The Dual Carboxymethyl Cellulase and Gelatinase Activities of a Newly Isolated Protein from Brevibacillus agri ST15c10 Confer Reciprocal Regulations in Substrate Utilization

Maiti

Samanta²,

Sahoo³

et al. 2017

Microb Physiol

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A protein showing endoglucanase-peptidase activity was prepared from a newly isolated bacterium (ST15c10). We identified ST15c10 as Brevibacillus agri based on electron-microscopic images and its 16S-rDNA sequence (GenBank accession No. HM446043), which exhibits 98.9% sequence identity to B. agri (KZ17)/B. formosus (DSM-9885T)/B. brevis. The enzyme was purified to homogeneity and gave a single peak during high-performance liquid chromatography on a Seralose 6B-150 gel-matrix/C-18 column. MALDI-TOF mass-spectrometry and bioinformatics studies revealed significant similarity to M42-aminopeptidases/endoglucanases of the CelM family. These enzymes are found in all Brevibacillus strains for which the genome sequence is known. ST15c10 grows optimally on carboxymethyl cellulose (CMC)-gelatin (40°C/pH 8-9), and also shows strong growth/carboxymethyl cellulase (CMCase) activity in submerged bagasse fermentation. The purified enzyme also functions as endoglucanase with solid bagasse/rice straw. Its CMCase activity (optimal at pH 5.6 and 60°C/K_m = 35.5 µM/V_max = 1,024U) was visualized by zymography on a CMC-polyacrylamide gel, which provided a strong band of approximately 70 kDa. The purified enzyme also showed strong peptidase (gelatinase) activity (pH 7.2/40°C during zymography on 6-12% gelatin/1% gelatin-PAGE (at approx. 70 kDa). The CMCase activity is inhibited by the metal ions Mn/Cu/Fe/Co (50%), Hg/KMnO₄ (100%), and by glucose or lactose (50-75%; all at 1 mM). The observed dose/time-dependent inhibition by Hg ions could be prevented with 2-mercaptoethanol. A comparison of the B. agri endoglucanase-aminopeptidase (ELK43520; 350 aa) with other members of the M42-family revealed the conservation of active-site residues Cys256/Cys260, which were previously identified as metal-binding sites. Regulation of the endoglucanase activity probably occurs via metal binding-triggered changes in the redox state of the enzyme. Studies on this type of enzyme are of high importance for basic scientific and industrial research.

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Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Cited by 20 publications

References 0 publications

Fecal carriage of ESBL-producing <em>Escherichia coli</em> in Egyptian patients admitted to the Medical Research Institute hospital, Alexandria University

Fecal carriage of ESBL-producing <em>Escherichia coli</em> in Egyptian patients admitted to the Medical Research Institute hospital, Alexandria University

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The Dual Carboxymethyl Cellulase and Gelatinase Activities of a Newly Isolated Protein from Brevibacillus agri ST15c10 Confer Reciprocal Regulations in Substrate Utilization

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