Plasmid Analysis Abstract: Plasmid DNA is the extrachromosomal DNA present in the bacteria. This plasmid DNA is able to carry the insert genes with it, and these genes are able to express themselves in the bacteria. When a gene is inserted into the Plasmid DNA, it is called Recombinant DNA…
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The blue plasmid DNA did not contain any of the restriction sites for Hind III or Sac I. Introduction Plasmids are the extra chromosomal DNA molecules which are mostly double –stranded, circular and covalently closed molecules, varying in size from 1 kb to 200 kb. (Sambrook and Russell 2001). They are found in many bacterial species. They replicate independently and use a variety of mechanisms to maintain their copy number. They contain the gene codes for the enzymes that are important for the bacterial hosts. The plasmids act as vectors in the molecular biology experiments. The vectors are the carrier DNA molecules into which the foreign gene of interest is inserted and expressed in the host. This DNA is now called recombinant DNA (Roberts and Murray 1976). This recombinant DNA is able to express the Foreign DNA in the bacteria. These vectors replicate inside the host cell along with the inserted DNA. These vectors are of two types: expression vectors (expression of the cloned gene to give the desired protein) and cloning vectors (produce millions of copies of cloned DNA) (Sambrook and Russell 2001). Restriction endonucleases are the enzymes that cut the DNA at the specific sequences. There are about 200 different restriction enzymes (Siwach and Singh 2007). ...
The restriction mapping is used to identify the plasmids. The number of DNA fragments and the size of the DNA fragments depend upon the action of the restriction enzyme (Kruezer and Massey 2008). These DNA fragments thus obtained are separated using the Agarose gel electrophoresis. Restriction mapping consists of three important steps. They are restriction enzyme digestion, agarose gel preparation and sample loading (Kruezer and Massey 2008). Results and Discussion: The nutrient agar plate was inoculated with E.coli, and the antibiotic discs were placed in the four quadrants. Figure1: Antibiotic profile against tetracycline in E. coli DH5alphaE:: pMTL84445 After inoculation at 37 degree Celsius for overnight, it was observed that the antibiotic disc of tetracycline had a clear zone. This indicates that the E.coli culture is resistant to kanamycin, chloramphenicol and ampicillin. There is very little sensitive to tetracycline. Figure 2: Antibiotic resistance profiling: Table 1a : Antibiotic resistance profiling of kanamycin control Kanamycin control E. coli DS941::pRRK Antibiotic disc Zone diameter in mm Chloramphenicol 30 Kanamycin 0 Tetracyline 10 Ampicillin 0 E.coli DS941::pRRK bacteria was found to be very sensitive to Chlormaphenicol and comparatively sensitive for Tetracycline antibiotics. The bacteria showed resistance to kanamycin and Ampicillin. Table 1b : Antibiotic resistance profiling of chloramphenicol control Chloramphenicol control: E. coli DS941::pAV35 Antibiotic disc Zone diameter in mm Chloramphenicol 0 Kanamycin 27 Tetracyline 32 Ampicillin 0 E. coli DS941::pAV35 bacteria were found to be very sensitive to Kanamycin and Tetracycline and resistant to Chloramphenicol and Ampicillin.
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Cloning entails the intensification of specific DNA segments, leading to the buildup of large amounts of similar DNA fragments, which may be utilized for numerous functions. Conventional molecular cloning uses tailored DNA cloning vectors, which are usually derived from spherical DNA molecules identified as bacterial plasmids.
They replicate independently and use a variety of mechanisms to maintain their copy number. They contain the gene codes for the enzymes that are important for the bacterial hosts. The plasmids act as vectors in the molecular biology experiments. The vectors are the carrier DNA molecules into which the foreign gene of interest is inserted and expressed in the host.
Many also contain a unique origin of replication and are autonomously replicated to high copy number within bacterial cells. Plasmids are frequently used for gene cloning purposes. Restriction enzymes are
ed cells are placed in liquid nutrient media after the heat pulse in order to start plasmid replication and expression to synthesize enough resistance enzymes to be able to survive when encountered with the antibiotic when plated onto the selective medium.
Tables have also been shown to represent the results either of the concentrations of the DNA of the plasmid and the genomic DNA concentration. Other results that have been tabulated are the transformation data for both the blue and white colonies. The
The resultant frequency of transformation was estimated at 1920 transformants/μg of DNA. This value was within the anticipated range of 800-7000 transformants/μg of DNA, thus the transformation process was successful.
The next step involved
This number was partitioned into 2 tubes; each tube contained 1.84 x 106 SW480 cells. This number of cells was adequate to go on with the RNA extraction process. However, it is good to note that the recommended starting point is 3 x 106 cells per
The conclusion from this study states that the transformation of bacterial cells requires a pre-exposure to Calcium Chloride in order to increase the chances of DNA uptake. Gene knock out is a possibility and the yeast cell proved that specific gene knock out and therapy can occur. The recombinant DNA technology has a revolution in molecular biology.
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