Abstract. Taq Polymerase Is Essential In Polymerase Chain

Polymerase Chain Reaction was run and the results were placed into an electrophoresis gel to visualize the PCR product. To determine the alleles in the gene, a digest enzyme, Fnu4HI, was placed into the DNA. Tasters would view a cleavage formed at a restriction site in at least one of the alleles, whereas non-tasters would not.

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

With two T7 promoters, the PCR product will be transcribed by the T7 polymerase. Therefore, a double stranded RNA (dsRNA) product is produced from the plasmid, once IPTG is added to activate the lac operator of T7 polymerase.

They were run at 1X 94ºC for 3 minutes, 30X at 94ºC for 30 seconds; 50ºC for 30 seconds; 72ºC for 45 second and 1X at 72ºC for 5 minutes. The PCR reactions took about 1 hour and 30 minutes to complete. The PCR products, were then purified by removing the leftover primers, nucleotides and salts. 250 µl of Buffer BB were added to Tube B and the mixture was pipetted into a spin column. The mixture was centrifuged for 30 seconds at room temperature. Then 2 cycles were completed at 30 seconds each with 200 µl of Buffer WB to remove any impurities. Then 25µl of Buffer EB were added to the tube to release the pure DNA and the mixture was centrifuged for 30 seconds. As the PCR reaction was running, a microscope slide was prepared from the live bacterial culture to observe the individual cells of the unknown bacteria and determine its

There are three steps in PCR, and they are; (1) Denaturing- DNA molecules are heated and separated into two single strands. (2) Annealing- A primer is used to start the process of building a new strand of DNA. (3) Extension- dNTPs are added to the reaction mixture to build a new complementary strand to the template strand. By using a primer a new strand grows in 5’ to 3’ direction and the template is 3’ to 5’ and they can be amplified by the original template.

2. McClean, Phillip. (1997). Cloning and molecular analysis of genes: Polymerase chain reaction (or PCR). Retrieved on December 6, 2014, from http://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/ clone9.htm. 3.

The method used in this experiment is the non- organic extraction where cell lysis solutions, proteinase K, isopropanol and ethanol was used. The functions of these solutions will be further discussed in the discussion.

A future experiment for site directed mutagenesis/PCR might involve a slightly increased amount of template DNA and primers in the PCR reaction or increased amount of PCR product that is being transformed, in case if the transformation of the plasmid did not work. Another possible change could be done to the PCR thermal cycles, by altering the denaturation and annealing temperatures to a reasonable degree. This is to ensure amplification of the desired plasmid DNA incorporating the desired mutation with accuracy at the appropriate locations within the DNA template. A future experiment for protein expression and purification should be done with further precision. And more care needs to be taken as to include all necessary antibiotics during transformation and expression, depending on the type of plasmids used in the experiment. This is essential to obtain the desired protein containing the appropriate genes to procced with further

Like DNA replication within an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the

Then 5 µL of each PCR sample was loaded onto a 2% agarose gel in the following order: Bioneer 25/100bp mixed DNA ladder, WT CCR5 plasmid, ∆32 CCR5 plasmid, cheek DNA, negative control. The gel was allowed to develop.

The components that were added to the PCR reaction is the template DNAs, which are the non-GMO sample, the test food sample, and the GMO positive sample. The rest of the items that were added were enveloped in the master mix, that consists of dNTPs, a buffer, primers, and Taq DNA polymerase. The dNTPs are deoxyribonucleoside triphosphate which are basically the nucleotides needed to elongate a DNA sequence. The buffer is important because it creates the correct pH and salt condition for DNA polymerase and because it also harbors magnesium ions in it, which serve as a cofactor for DNA polymerase. Taq DNA polymerase is vital because it is a heat stable enzyme derived from the bacteria Thermus Aquaticus that is able to withstand the high temperatures

Polymerase chain reaction (PCR) is the most common form of DNA analysis (National Institute of Justice). PCR was invented in 1983 by Dr. Kary Mullis. Mullis was working on a project and could not complete it. He said he needed a way to increase the concentration of a specific gene of interest (Oswald, 2017). As he was driving home, he thought that "Using two opposed primers, one complimentary to the upper strand and the other to the lower, then performing multiple cycles of denaturation, annealing and polymerization he could amplify the piece of DNA between the primers" (Oswald, 2017). This is how PCR came about. According to Garibyan and Avashia (2013), "PCR is a simple, enzymatic assay, which allows for the amplification of a specific DNA fragment from a complex pool of data" (p. 1). PCR is a good technique to use because only trace amounts of DNA are needed to generate enough copies to be analyzed. The presence of template DNA, nucleotides, primers, and DNA polymerase are required for every PCR test. It is important to understand what these components are. There are four nucleotides found in DNA and they are adenine, thymine, cytosine, and guanine. Primers are important because this specifies the exact DNA product that must be amplified. Primers are short DNA fragments that have a defined sequence that is complementary to the target DNA that is to be detected

The initial Sanger sequencing method has been administered to several significant improvements and developed remarkably over three decades. Even though cloning of DNA fragments into plasmid vector was required in Sanger sequencing, the application of polymerase chain reaction (PCR) (Saiki et al., 1988) for the amplification of specific DNA fragment in vitro has extensively

Background: Polymerase Chain reaction (PCR) is an in vitro process that enables exponential amplification of the define target DNA sequence. The process involves three steps: denaturation, annealing and elongation. In the first step, the reaction mixture containing target

Polymerase chain reaction (PCR) can be termed as an enzymatic, molecular “xeroxing” process in which a particular region of DNA is replicated several times to yield thousands of copies of a specific sequence. The process of PCR employs a precise pattern of heating and cooling of samples in a thermal cycler, over 30 cycles.