Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4090 ◽  
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Plasmid Dna ◽  
Alkaline Lysis

scholarly journals Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Kuan Yao ◽  
Narjol González-Escalona ◽  
Maria Hoffmann
Keyword(s):  
Antibiotic Resistance ◽  
Single Molecule ◽  
Plasmid Dna ◽  
Copy Number ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Fast Method ◽  
Alkaline Lysis ◽  
Low Copy Number ◽  
Long Read

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.

Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation

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

scholarly journals Molecular characterization of covalently closed circular plasmid DNA in local Streptomyces strains isolated from soil with detection of antibiotic production and antibacterial activity

2019 ◽  
Vol 10 (3) ◽  
pp. 2142-2152
Author(s):  
Suad A Al-Hilu ◽  
Wisam H Al-Shujairi
Keyword(s):  
Antibacterial Activity ◽  
Yeast Extract ◽  
Plasmid Dna ◽  
Antibiotic Production ◽  
Selective Medium ◽  
Malt Extract ◽  
Biochemical Tests ◽  
Alkaline Lysis ◽  
Mannitol Salt Agar ◽  
Streptomyces Species

Various of Streptomyces species have two kinds of plasmids, circular plasmids (8 to 31 kb) and linear plasmids (12 to 1700 kb). Covalently closed circular (CCC) plasmids are profuse in the genus of Streptomyces and involved in production and resistance of antibiotics by genetic controlling. We collected fifty clinical soil samples from different regions in Al-Najaf Al-Ashraf province/Iraq. The samples included five from Al-Ghadeer Quarter, five from Al-Karama Quarter, 10 from Kufa University, five from Al-Ameer Quarter, four from Al-Forat Quarter, 10 from North Quarters and eleven from desert roads in Al-Najaf. Diluted samples were cultured on Yeast extract Malt extract (YEME) agar medium as a selective medium; then the presumptive Streptomyces colonies were subcultured on Tryptone Yeast extract (TYE) agar, then incubation at 37ᵒC for 7 days. Seven biochemical tests for identification of Streptomyces isolates these are: Catalase test, Oxidase test, Urase test, Kligler Iron Agar test (KIA), Simmon᾽s Citrate test, addition to MacConkey agar test and Mannitol Salt agar test. Five antibiotic discs were used for detection of antibiotic sensitivity of the Streptomyces isolates; these are: Tetracycline, Gentamycin, Vancomycin, Ampicillin, Erythromycin. The sensitivity of the antibiotics was observed by recorded the diameter of inhibition zone around the discs. Two test bacteria (Staphylococcus aureus and E. coli) were used for the determination of antibacterial activity. Plasmid isolation was done by the alkaline lysis method. This method is characterized by the rapid isolation of DNA from Streptomyces. Then, detection of Plasmid DNA occurred by using agarose gel electrophoresis.

scholarly journals Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol

BioTechniques ◽  
2021 ◽  
Author(s):  
Viiu Paalme ◽  
Mart Speek
Keyword(s):  
Quality Control ◽  
Column Chromatography ◽  
Plasmid Dna ◽  
Fractional Precipitation ◽  
Alkaline Lysis ◽  
Bacterial Extract ◽  
Total Rna ◽  
Spin Column ◽  
Commercial Kits

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli

2013 ◽  
Vol 246 (11) ◽  
pp. 861-867 ◽  
Author(s):  
Saša Haberl ◽  
Marko Jarc ◽  
Aleš Štrancar ◽  
Matjaž Peterka ◽  
Duša Hodžić ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Alkaline Lysis ◽  
Electric Pulses
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