Construction & Diagnostic of Recombinant DNA Plasmid - Research Paper Example
The experiment established that the competency of cells increased greatly.Â In the next experiment, running PCR provided me with an amplified His3 gene which is used in subsequent steps to transform haploid yeast. Transformation occurred through homologous recombination. The experiment proved that specific integration does occur although cases of non-specific integration are rampant. In the next experiment, I constructed a sub-clone of the HIS3 gene and inserted it into a plasmid pSP72 making a recombinant plasmid which I used to transform bacterial cells. In order to determine whether the integration was successful, I extracted the plasmid to analyze if the inserted gene was present. However, I established that integration had not been successful. Introduction One of the laboratory techniques that help in understanding the basics of knocking out genes is the standardized procedure of replacing the ADE2 gene responsible for adenine biosynthesis with HIS3 that is responsible for one of the steps in histidine amino acids. The procedure involves production of a hybrid polymerase chain reaction (PCR) as the first step. The hybrid product constitute of the vector DNA template and the primers of choice. Running of the PCR major steps produces the hybrid product. Agarose gel electrophoresis helps in determining whether PCR amplification occurred. The next step involves the transformation of yeast cells with the hybrid PCR product. The last step involves analysis of r4esults and morphology of transformed yeast cells. Other experiments involve transforming competent Escherichia coli cells with the use of a plasmid as a vector. This transformation follows the recombinant DNA technology protocol. The general procedure starts with digestion of plasmid DNA and template DNA of interest with restriction enzymes to generate DNA fragments with sticky ends. The second step involves ligation of the DNA fragments using DNA ligase, forming a recombinant plasmid. The next step involves insertion of the recombinant plasmid into the competent bacterial cells. The final step involves plating on appropriate media and selection of transformed cells. In addition, performing a backward procedure of isolating the plasmid from the transformed cells verifies insertion at the right locus. Laboratory 1: Transformation of Competent Bacteria Objective: Introduction of Plasmid DNA into E. coli cells and determination of transformation efficiency Materials and Reagents: Plasmid DNA Gene of interest SOC media LB-amp media Procedure: The protocol preferred was the High-Efficiency Transformation Protocol. However, a variation occurred with 2Âµl of plasmid DNA. The high efficiency transformation protocol requires thawing of competent cells in ice for about ten minutes. The next step involves transfer of 50Âµl of the cells to a transformation tube using a micropipette. Adding of 2Âµl of plasmid DNA into the tube followed. The next step involved placing the mixture on ice for 20 minutes. Next, exposure of cells to heat shock at 42?C occurs, a process lasting 35 seconds. Following this was adding the right amount of SOC media to the cells. After this, incubation at 37C? for 40 minutes and subsequent vigorous shaking followed. Plating of the cells in LB-amp media and overnight incubation at 37?C was the last step. In the first experiment, plating of the concentrated cell mixture without dilution occurred. In subsequent trials, there was dilution of cell solution at different dilution ratios.
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