Isolation of plasmid DNA from E. coli (Alkaline lysis method) v1

protocols.io ◽  
2016 ◽  
Author(s):  
Anna Behle
Keyword(s):  
2020 ◽  
Vol 14 (1) ◽  
pp. 63-77 ◽  
Author(s):  
Manzar Alam ◽  
Mohd Imran ◽  
Syed Sayeed Ahmad

Aim: Our exploration work has uncovered the different anti-toxin/metal tolerance and patterns against the heavy metal resistant coliform microscopic organisms from the aquatic waste of the hospital. It might give new routes for the treatment of irresistible ailments particularly by coliform and critical for hazard evaluation as well as hazard management associated with the effluents of the hospital. Background: The higher use of pharmaceuticals, Radionuclides, and other antimicrobial solvents are the major source of metals in hospital wastewater. The hospital aquatic environment has a high content of both organic and inorganic matter with living organisms. Bacteria can resist an antimicrobial agent by producing extracellular enzymes that eliminate antibiotics and metal toxicity. In this study, we covered the existing patent literature in this area. New patents in the areas of topically applied antibiotics and agents that can potentiate the achievement of existing antibiotics may extend their helpful lifetime. Methods: Samples were collected from three different Departments of King George Medical University, Lucknow during the month of December to May (2015-16). Isolation and metal tolerance of coliform isolates were done on metal amended plates. The antibiotic sensitivity test was done by disc diffusion method. The plasmid DNA of bacterial isolates was done by the alkaline lysis method. The conjugation study was also performed in wastewater as well as a nutrient medium. Results: Maximum isolates demonstrated their MICs at 400, 800 and 1600 μg/ml against all the metals, respectively. The high level of resistance was observed against Methicillin (88.32%, 80.60%) followed by penicillin (75%, 76%), Cephradin (59.52%, 28.84%) and least to Gentamycine (1.92%, 5.76) in E. coli and Enterobacter, respectively. Of 70%, 78% E. coli and Enterobacter isolates produce beta-lactamase activity. Six amino acid residues namely, Glu104, Tyr105, Asn132, Asn170, Ala237, and Gly238 of the beta-lactamase were found in the common interaction with the selected drugs. Plasmid DNA size ranged between 48-58.8 kb. The conjugation experiments showed a higher transfer frequency (5.5×10-1 and 3.6×10-1) rate among antibiotics and metals tested. Conclusion: The finding of this study presents a potential health problem as the predominant coliform species have increasingly been associated with outbreaks of hospital infections. It is recommended that hospital waste must be properly treated before its release into the environment.


2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.


2021 ◽  
Vol 12 ◽  
Author(s):  
Kuan Yao ◽  
Narjol González-Escalona ◽  
Maria Hoffmann

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.


2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot3901 ◽  
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  

2019 ◽  
Vol 6 (2) ◽  
pp. 54
Author(s):  
Folarin ◽  
Nesbeth ◽  
Ward ◽  
Keshavarz-Moore

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.


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