What is Plasmid Prep?Isolation of Plasmid DNA from E. coli is a common routine in research laboratories to purify a specific sequence, since they can easily be purified away from the rest of the genome. You will perform a widely‐practiced procedure that involves alkaline lysis of cells. This protocol often referred to as a Plasmid "mini‐prep," yields fairly clean DNA quickly and easily.

Background: Most of molecular biology studies depend on making gene constructs. Although commercial Plasmids are widely used for this purpose, sometimes due to the nature of the restriction sites or need of multiple subcloning, usual restriction sites available in original multiple cloning sites (MCS) of the Plasmids could not be easily used, if not impossible at all. Here, we report an easy and fast method to construct suitable Plasmid with a new MCS for constructing a 16kb gene targeting Plasmid. Methods: Firstly, the suitable MCS was designed by studying the sequence of desired gene fragments in Gene runner software. Two partial complementary 74 base long oligonucleotides were designed and constructed to make this MCS. The original pUC19 MCS was replaced with the new one by enzymatic digestion of the Plasmid and removal of the MCS, followed by adding the two complementary oligonucleotides and ligating the construct and transforming it into Ecoli TOP10 F'. The new Plasmid was then purified and sequenced by M13 forward and reverse primers. Findings: Synthesis of two 74 base polynuclotides was successful, and these polynucleotides formed a double stranded fragment which was successfully inserted between HindIII-EcoRI sites of pUC19. Analysis of intermediate step results showed successful progress of cloning reaction. Final analysis of the Plasmid by restriction analysis and sequencing the MCS confirmed authenticity of the new Plasmid. Conclusions: The method described here is a fast and easy way to make suitable Plasmid out of commercially available Plasmids. This process can considerably decrease the time and cost of Plasmid construction. Availability of suitable restriction sites in proper order makes it possible to directionally clone the desired gene fragments which is more efficient and excludes screening steps for the right direction of the fragments. The Plasmid reported herein is specifically tailored to insert different fragments of a beta-globin gene targeting construct.  

Background: Chitosan, a biodegradable polymer with no toxicity, has also been targeted as a DNA vector. In this study, the effects of lysozyme on chitosan / Plasmid nanoparticles were investigated. Methods: In this study, the gelation method was used to make nanoparticles. After studying the percentage of Plasmid loading in chitosan nanoparticles and also the Nanoparticle properties, the nanoparticles were treated with lysozyme and the effects of enzymatic digestion were investigated. Results: The results of this study showed that the percentage of Plasmid loading in the nanoparticles was 100%. The size, dispersion index, and zeta potential of the nanoparticles were 173 nm, 0. 292 and 10. 08 mV, respectively. the results of enzymatic digestion showed that the solution containing lysozyme at concentration of 10 µ g / ml and enzymatic power of 100 u / ml after 4 hours was able to partially digest chitosan nanoparticles but did not completely release the Plasmid. but after 24 hours, it releases the Plasmid completely from the nanoparticles. Conclusion: By reviewing the results of this study and comparing it with the findings of other studies on chitosan, it is found that in addition to optimal conditions for lysozyme enzyme function, the time factor is also important for lysozyme function in complete release of DNA from chitosan / Plasmid nanoparticles.

Background: Antibiotic resistance is a problem around the world. Some bacteria are resistant to these medicines. Medicinal plants and derivatives are good source for therapy of these diseases.Objective: The curing of four medicinal plant extracts (Thymus caramanicus (T.c), Dracocephalum polychaetum (D.p), Adianthum capillus-veneris (A.c), Viola tricolor (V.t)) on Plasmid bearing strains of klebsiella pneumoniae resistance to multiple antibiotics were investigated.Methods: Klebsiella pneumoniae resistance to multiple antibiotics were isolated from hospital infectious samples. Alcoholic and maceration extraction were carried out from leaves, flowers or roots of the above plants. Minimum inhibitory concentrations (MIC) of each plant extract was measured. At the same time the sub inhibitory concentration (SIC) and minimum bactericidal concentration (MBC) activity of each plant extract was carried out in order to cure Plasmids from Plasmid bearing strains. The sensitivity of the strains treated with SIC of each plant extracts to ampicillin, amoxicillin, cefotaxime, ceftizoxime, vancomycin has been measured.The morphological changes and capsule staining of klebsiella pneumoniae has been investigated with fluorescent microscope.Results: Minimum inhibitory concentrations (MIC) of each plant extract indicated that all of them have anti microbial activity and were able to inhibit the growth of resistant strains of kleb.pneumoniae. Furthermore the T.c and A.c extract had highest anti microbial activity as compared to other plant extracts.Kleb. pneumoniae cells treated with T.c and A.c extract, the cells aggregated and change their morphology, similarly, production of capsule was also lost. In addition gram stain properties of bacteria has been changed. That is the high mutagenic properties of these plant extracts on gene or gene expression in capsule modulation.The agarose gel electrophoresis (0.7%) of the treated cells and untreated one and observation of the gel with U.V. gel documentation system indicated that there were any physical loss of Plasmid band on the gel.Conclusions: From above data it could be concluded that the above plants extract though they have antibacterial activity they didn't exert any curing effect on the Plasmid bearing microorganisms, but antibacterial effects of T. c and A. c extracts are considerable.

The curing of five medicinal plant extracts (Satureia hortensis, Gly cyrrhiza glabra, Teucrium Pallium, Myrtus Communis, Eukalypetus globolus) on Plasmid bearing strains of klebsilla pnenmoniae resistance to multiple antibiotics were investigated.Alcoholic and maceration extraction were carried out from leaves, fruits or roots of the above plants. Mimimum inhibitory concentrations (MIC) of each plant extract indicated that all of them have antimicrobial activity and were able to inhibit the growth of resistant strains of kleb. pneumoniae. Furthermore the eucalypetus extract had highest antimicrobial activity as compared to other plant extracts (avrage MIC 0.1mg/ml). At the same time the subinhibitory concentration (SIC) and minimum bactricidal conctration (MBC) activity of each plant extract was carried out in order to cure Plasmids from Plasmid bearing strains. Curing experiments were performed from SIC tubes which then diluted serially (10-2, 10-4, 10-6, 10-8) and 100 well isolated colonies were then master plated with sterile tooth pikes. The sensitivity of the strains treated with SIC of each plant extracts to ampicillin amoxicillin, cefotaxime, and ceftizoxime indicated that there were no change in MIC of antibiotics compared to unexposed one.The morphological investigation with flouresent microscope (Nikon, optiphota-2) and capsule staining indicated that when cells treated with Myrtus Communis extract, the cells aggregated and change their morphology, similarly, production of cupsule was also lost. The agarose gel electrophoresis (0.7%) of the treated cells and untreated one and observation of the gel with U.V. gel documentation system indicated that there were any physical loss of Plasmid band on the gel.Similarly, the synergistic action of plant extract and antibiotics were investigated and it was found that these was no synergy between the above plant extracts and antibiotics (MIC remained unchanged). From above data it could be concluded that the above plants extract though they have antibacterial activity they did not exert any curing effect on the Plasmid bearing microorganisms isolated from hospitals in kerman Iran.

Genetic evidences have shown that the rhizobium bacteria nodulate the legume plants because of nod, sym and fix genes. Almost all members of rhizobaceae family harbor large Plasmids, which are highly variable in number and size. Representative of nif, fix and nod genes have been located on the symbiotic Plasmids of different rhizobium species. Therefore, the size and numbers of Plasmids of bacterial isolates (by the Plasmid profile technique) could be used as a diversity index in ecological studies. In this investigation, the diversity of 196 isolates of sinorhizobium sp isolated from Hamada soils was evaluated by using Plasmid profile technique. The results showed that the number of Plasmids among all isolates varied from 1 to 4 and totally 13 different Plasmids were identified. The size of Plasmids varied from 50 to 200 kb. Isolates with 1, 2, 3 and 4 Plasmids formed 63, 21, 13 and 2 percentage of the population. Isolates of sinorhizobium with 2 and 3 Plasmids were clustered into 8 and 15 groups, respectively, based on Plasmid patterns. Four isolates which contained 4 Plasmids were grouped in four different clusters and finally all isolates of Sinorhizobium (196) were grouped in 28 different groups.