scholarly journals The Nav channel bench series: Plasmid preparation

MethodsX ◽  
2014 ◽  
Vol 1 ◽  
pp. 6-11 ◽  
Author(s):  
Daniel H. Feldman ◽  
Christoph Lossin
Keyword(s):  
Plasmid Preparation

scholarly journals Fast, non-toxic, and inexpensive n-butanol preparation of recombinant plasmids

2000 ◽  
Vol 23 (2) ◽  
pp. 299-300
Author(s):  
Jürgen Brieger ◽  
Eberhard J. Weidt ◽  
Jochen Decker
Keyword(s):  
Toxic Chemicals ◽  
Recombinant Plasmids ◽  
Plasmid Preparation

Various commercial and non-commercial plasmid preparation protocols are currently available. However, the kits are expensive and many of the protocols contain toxic chemicals. Here we present a novel, optimized and, therefore, very advantageous plasmid preparation protocol using n-butanol. The preparation can be performed quickly and no toxic chemicals are used, at overall costs of about one cent per plasmid preparation.

scholarly journals Two step PCR method to exchange the resistance cassette of a vector

2021 ◽  
Author(s):  
Nils Cremer ◽  
Anne Diehl
Keyword(s):  
Antibiotic Resistance ◽  
Agar Plate ◽  
Easy Method ◽  
Antibiotic Selection ◽  
E Coli ◽  
Target Vector ◽  
Pcr Product ◽  
Plasmid Preparation ◽  
Pcr Method ◽  
Dna Template

AbstractFor co-transformation of two plasmids, both have to possess different antibiotic selection markers. If that is not the case, normally the gene of interest (GOI) is subcloned into another vector. Here we introduce a fast and easy method to exchange the antibiotic resistance cassette (ARC) in only two PCR steps.Method SummaryTo shuttle the antibiotic resistance cassette (ARC) from one vector to another, one can amplify the ARC of interest and use the resulting PCR-product as a primer pair for the next amplification step. Simply remove parental DNA template by DpnI digestion, transform PCR product directly in E. coli cells, select transformants on an appropriate agar plate and isolate target vector by plasmid preparation.

scholarly journals Automated Filtration-Based High-Throughput Plasmid Preparation System

1999 ◽  
Vol 9 (5) ◽  
pp. 463-470 ◽  
Author(s):  
Masayoshi Itoh ◽  
Tokuji Kitsunai ◽  
Junichi Akiyama ◽  
Kazuhiro Shibata ◽  
Masaki Izawa ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Microtiter Plate ◽  
Linear Process ◽  
Alkaline Lysis ◽  
Filtration Method ◽  
Cell Harvesting ◽  
Automated Processing ◽  
Plasmid Preparation ◽  
Plasmid Purification

Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing.

p53 Plasmid Preparation and Techniques for Analysis of Gene Transfer and Expression

2003 ◽  
pp. 207-220
Author(s):  
Ragai R. Mitry ◽  
Michael D. Kelly ◽  
Jian Zhao ◽  
Satoko Negishi ◽  
Marc R. Mansour ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Plasmid Preparation

scholarly journals Antibiotic Resistance Profile and association with Integron Type I among Salmonella Enterica Isolates in Thailand

2020 ◽  
Vol 14 (4) ◽  
pp. 2383-2397
Author(s):  
Nartlada Onvimol ◽  
Phanita Chankate ◽  
Yuvadee Mahakhunkijcharoen ◽  
Thareerat Kalambaheti
Keyword(s):  
Gene Cassette ◽  
Acid Synthesis ◽  
Chicken Meat ◽  
Type I ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
Antibiotic Resistance Profile ◽  
Class 1 ◽  
Lactam Antibiotic ◽  
Plasmid Preparation

Salmonella infection is the second most common cause of diarrhea in Thailand; however, the data on antimicrobial resistance is limited. There were137 Salmonella strains, isolated from patients and 126 strains isolated from chicken meat, collected from Nonthaburi, Thailand during 2002. The top five serotypes of patients isolates were Enteritidis (22%), Typhimurium (11%), Weltevreden (8.8%), Rissen (8%), and Choleraesuis (6.6%) while the top five serotypes of chicken meat isolates were found as follows: Schwarzengrund (11.91%), Hadar (11.11%), Rissen (8.73%), Amsterdam (7.94%), and Anatum (7.94%). Salmonella strains were most resistance to the class of antibiotics that act as inhibitor to nucleic acid synthesis such as antifolates group (Trimethoprim;SXT) and fluoroquinolones (Nalidixic acid; NA, Ciprofloxacin; CIP),while the β lactam antibiotic was more effective, i.e. the 3rd gen cephalosporin (Ceftazidime; CAZ, Cefotaxime ; CTX), Monobactam (Aztreonam; ATM) and carbapenams group (Imipenem; IMP, Meropenem; MEM). The role of class I integron element in transmission of the resistance gene was revealed by detection the gene cassette associated with a class 1 integron in plasmid preparation among 80% of the isolated strains. The gene cassettes containing resistant genes of dhfrA12 (resistant to trimethoprim) and aadA2 (resistant to streptomycin and spectinomycin), were detected more frequently in the resistant strains. These gene cassettes were likely to be transmitted via plasmid, as it could not be detected in genomic DNA.

Lysis Buffer for Plasmid Preparation

2013 ◽  
Vol 2013 (2) ◽  
pp. pdb.rec073619-pdb.rec073619
Keyword(s):  
Plasmid Preparation ◽  
Lysis Buffer

scholarly journals Characterization and Nucleotide Sequence of a Klebsiella oxytoca Cryptic Plasmid Encoding a CMY-Type β-Lactamase: Confirmation that the Plasmid-Mediated Cephamycinase Originated from the Citrobacter freundii AmpC β-Lactamase

1999 ◽  
Vol 43 (6) ◽  
pp. 1350-1357 ◽  
Author(s):  
Shang Wei Wu ◽  
Kathrine Dornbusch ◽  
Göran Kronvall ◽  
Mari Norgren
Keyword(s):  
Nucleotide Sequence ◽  
Sequence Similarity ◽  
Klebsiella Oxytoca ◽  
Open Reading Frames ◽  
Citrobacter Freundii ◽  
E Coli ◽  
Plasmid Preparation ◽  
High Level ◽  
Rna I

ABSTRACT Plasmid pTKH11, originally obtained by electroporation of aKlebsiella oxytoca plasmid preparation intoEscherichia coli XAC, expressed a high level of an AmpC-like β-lactamase. The enzyme, designated CMY-5, conferred resistance to extended-spectrum β-lactams in E. coli; nevertheless, the phenotype was cryptic in the K. oxytocadonor. Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 β-lactamase (bla CMY-5). Thebla CMY-5 product was similar to the plasmidic CMY-2 β-lactamase of K. pneumoniae and the chromosomal AmpC of Citrobacter freundii, with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2. bla CMY-5 was followed by the Blc and SugE genes of C. freundii, and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome ofC. freundii; these results confirmed that C. freundii AmpC was the evolutionary origin of the plasmidic cephamycinases. In the K. oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63. Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible. Incompatibility of the two plasmids might explain the cryptic phenotype ofbla CMY-5 in K. oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.

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