Plasmid analysis

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.