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Essentially, Luria broth defines a a bacteria culture media that with high nutrients used in laboratories. In addition, isolation of…
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Download file to see previous pages 180). The latter permits replication of plasmid in host cells while drug-resistance gene is essential in allows growth of host cells by destruction of antibiotics especially carbenicillin.
The use of restriction enzymes, in obtaining cloning sites through cleaving of the vector acts as the initial step in insertion of foreign DNA (Seviour and Nielsen, 2010, p. 365). Sticky ends especially single-stranded ends can results from cleavage of palindromic sequence GAATTC by EcoRI (Russell et al., 2013, p. 393). The resultant single-stranded ends have hybridization ability with similar EcoRI pieces of DNA. Using the principle, scientists take sequences of foreign DNA for cloning and mix them with cleaved vectors after digestion with EcoRI (Brown, 2013).
After hybridization of the plasmid and foreign DNA through the sticky ends, the next step involves sealing with phosphodiester linkages to form recombinant plasmid. DNA ligase enzyme remains imperative in the sealing process. Consequently, the aforementioned replication origin, resistance gene, and DNA fragment remains present on the newly created recombinant plasmids that collectively forms circular library. The inherent recombinant plasmids have each of them possessing unique foreign DNA fragment.
Subsequent stage involves addition of E. Coli bacteria that acts as host cells to the recombinant plasmids. At this stage, the study has made the cells permeable to DNA through treatment with CaCL2. Some cells resist taking recombinant plasmids while others do through a process known as transformation.
Thereafter, the researcher pours the E,Coli cells into an antibiotic carbenicillin plate nutrient agar. It is important to note that only cells that have resistance to carbenicillinin antibiotic would grow in the agar as opposed to the rest. Growth and multiplication of the aforementioned cells remains possible at 37 ...Download file to see next pagesRead More
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