Via Polymerase Chain Reaction and Cloned
Isolated DNA from mouse, plants, and plasmid DNA were used for Polymerase Chain Reaction (PCR) for DNA amplification. The purpose of this experiment was to study the success rate or optimization of PCR of DNA, using both manual and kit methods. This set of experiments gives an insight to the relative difficulties associated with the optimization of a variety of DNA/primer paired samples. Through this experimentation, researchers were successfully able to assess the issues and difficulties in both the manual and kit optimizations that were performed. Through this report, step by step methods used to achieve viable PCR product are discussed, along with different methods of trouble shooting and experiences through out the experimentation. The PCR product’s amplified DNA was then used for insertion into a plasmid vector system, which too resulted in various range of consequences and difficulties. Hence, the success rate and difficulties are viewed and analyzed through out this paper as experimenters try to achieve maximal desired PCR products associated with each protocol used.
Polymerase Chain Reaction (PCR) is a technique used in molecular biology and biotechnology, to amplify a small amount of DNA in a reaction. This simple and inexpensive technique is a fundamental tool that focuses on the segment of given DNA and copies it over. Today, PCR is used in every day laboratories to study diseases, identify bacteria or viral DNA, and trace criminals.
To conduct a PCR reaction, a few critical elements are needed, such as: polymerase, primers, nucleotides, buffers, and the most crucial, the template that needs to be amplified. DNA polymerase, acquired from Thermis aquaticus, is the most important enzyme in this reaction, as it adds complementary bases to the template and binds to the region that needs to be amplified. Primers are designed to anneal to the template, at their specific annealing temperature. Nucleotides are the building blocks of all DNA molecules, and DNA polymerase collects and binds them to the ends of the primers for extension of a new strand which is complementary to the cold stand.
Through the PCR method of heating and cooling the reaction to a set of temperatures in a series of steps, DNA is able to exponentially grow to a desired product. In this lab, DNA isolated from mouse liver, tissue, and plasmid DNA extracted from E.coli was used to amplify the part of the genes using PCR application. Afterwards, the successful PCR products were used for cloning and restriction analysis, which was the ultimate goal.
With successful PCR products, there are a number of experimental procedures experimenters can do with PCR products. In this case, the ultimate goal was to insert PCR products into a plasmid vector engineered to act as a cloning vector for small DNA segments. Blue-white screening process is a technique used for the determination of the successful ligations into the vector. With successful insertion, the pigmentation of the cells change to white, indicating insertion was successful. The change in color occurred because the insertion site for the DNA fragment of interest falls into the coding for Lacz, therefore interrupting the cell to produce Beta-galactosidase. When lactose was successfully cleaved, the colonies appear blue on an X-Gal plate. However, if lactose was not cleaved, the cells were white. As the focus of this experiment was to examine and compare the different methods of PCR optimization, as well as assess the difficulties involved, the research took place over a span of 5 to 6 weeks.
Materials and Methods
PCR is a relatively simple and inexpensive tool that is used to focus in on a segment of DNA and copy it billion of times. A DNA template sample that contained the target sequence was used to replicate. For the...