scholarly journals Preparing low-copy number plasmid DNA using the ZymoPURE II Plasmid Maxiprep Kit

BioTechniques ◽  
2018 ◽  
Vol 65 (6) ◽  
pp. 365-367
Keyword(s):  
Plasmid Dna ◽  
Copy Number ◽  
Copy Number Plasmid ◽  
Low Copy Number

scholarly journals Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

1982 ◽  
Vol 150 (3) ◽  
pp. 1234-1243 ◽  
Author(s):  
W Firshein ◽  
P Strumph ◽  
P Benjamin ◽  
K Burnstein ◽  
J Kornacki
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Copy Number ◽  
Membrane Complex ◽  
Copy Number Plasmid ◽  
Low Copy Number

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.

scholarly journals Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
Author(s):  
Satoshi Tomita ◽  
Kenji Tsuge ◽  
Yo Kikuchi ◽  
Mitsuhiro Itaya
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Positional Cloning ◽  
Deletion Mutant ◽  
Essential Gene ◽  
Transfer System ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

scholarly journals A vector for studying plasmid stability functions in Streptomyces

1988 ◽  
Vol 51 (1) ◽  
pp. 71-74 ◽  
Author(s):  
Kevin Kendall ◽  
John Cullum
Keyword(s):  
Copy Number ◽  
Stability Region ◽  
Selectable Marker ◽  
Plasmid Stability ◽  
Stability Function ◽  
Melanin Production ◽  
Stability Functions ◽  
Tyrosinase Gene ◽  
Copy Number Plasmid ◽  
Low Copy Number

SummaryWe constructed a cloning vector (pMT603) based on the low copy number plasmid SCP2*. pMT6O3 is unstable because it lacks the SCP2* stability region and carries the selectable marker thiostrepton-resistance and a tyrosinase gene which results in melanin production. This allows easy testing of plasmid stability and we demonstrated its usefulness by cloning a plasmid stability function.

scholarly journals A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

2012 ◽  
Vol 54 (2) ◽  
pp. 504-514 ◽  
Author(s):  
Giyoun Na ◽  
Andrew Wolfe ◽  
CheMyong Ko ◽  
Hyesook Youn ◽  
Young-Min Lee ◽  
...  
Keyword(s):  
Copy Number ◽  
Copy Number Plasmid ◽  
Low Copy Number
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