Polymerase Chain Reaction: Lab Technique

Before it undergoes PCR the DNA extraction has a few steps to be done first, which is crucial to allow for amplification to begin, it needs to undergo cell lysis and denature the proteins, which then is purified (Tracey, 2017). The extracted DNA was then put into NaOH, a strong base and heated to 95 degrees Celsius, which leads to breaking down the hydrogen bonds between the nitrogenous (Tracey, 2017). Once all the previous steps have been completed, the PCR reaction can commence after mixing with primers, Taq DNA polymerase, reaction buffers and dNTPS. The Thermocycler was utilized to facilitate PCR making it faster, more reliable and cheaper (Tracey, 2017). The sample will undergo 35 amplification cycles, producing 3.44x1010 target sequences [ 235= 3.44x1010

(PCR), which isolates small fragments of DNA that have a high degree of variability from

PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. Steps of the PCR cycle are shown in below figure.

A PCR tube containing a Ready-To-Go™ PCR Bead was supplemented with 22.5 microliters of a solution containing TASR38-specific primers. 2.5µL of the mixture were added to the primer mixture, and the sample was stored in ice until the entire group had finished the process up to this point. The entire group’s samples of DNA were denatured for 20 seconds at 95°C, then incubated for 20 seconds at 64°C so that the primers could anneal, then incubated at 20 seconds at 72°C, and then polished for five minutes at

Since the temperature optimum for the DNA polymerase to act is 72° C, the reaction is heated to that temperature. DNA polymerase lengthen the primers by attaching more nucleotides onto the primer in a sequential manner, using the target DNA as a template.

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

The primary purpose is to identify a genetic marker or study the function of a specific gene. There are three steps involved in this process which are as follows: denaturation, annealing and elongation. Denaturation involves heating the DNA to agitate the hydrogen bonds, and annealing allows the temperature to be lowered so that the primers can be “annealed” to the single-stranded DNA template. The last step requires DNA polymerase to synthesize a new strand of DNA that is complementary to the RNA strand in the 5’ to 3’ direction (Amplifying DNA: The Polymerase Chain Reaction, 2016). The forward and reverse primers are needed to start the replication process by providing the appropriate nucleotides to the new strand. On the contrary, sanger sequencing makes copies of a target DNA, and the the DNA strand that will be sequenced is separated into two strands, so they can be copied through chemically altered bases. The altered bases cause the process of copying to terminate each time a particular letter is added to the growing DNA chain, which happens to all four bases until the fragments are put together to reveal the original sequence of the original DNA. The aforementioned processes are thoroughly explained to give an overview of the steps involved in providing the end products of the experiment, so an individual can manually decipher

PCR works by denaturing the double-stranded DNA and annealing the primers to the newly-made single-stranded DNA, leading to the extension/elongation of the DNA by a polymerase that attaches to the primer/DNA strand. The PCR reaction strums through a handful of temperature cycles to maximize each step and the amount of product.

This process consists of three major steps which are denaturing, annealing, and elongation. The cells are lysed before the denaturation step in order to obtain chromosomal DNA. In order to denature the DNA, or separate the two strands, the DNA is heated to 95 degrees Celsius. Once the denaturing is complete, the PCR tube is cooled to approximately 55 degrees Celsius during the transition to the annealing step. During this step, there are two primers: 27F and 1492R, which bind to the 16S gene on the strands of DNA. A high molar ratio of primers is used to ensure that there is attachment between the selected region of DNA and the primers as well as to ensure that the primers will hybridize. After the annealing step, elongation happens at around 75 degrees Celsius. A Master Mix is added during this step, which contains Taq polymerase and dNTPs. In order to replicate the sequence, Taq polymerase uses the dNTPs. Since the primer is on the 5’ end of the strand, the elongation is happening in the direction of the 3’ strand and the elongation is indefinite and these

Our materials for the extraction of DNA included: the screwcap tubes, capless tubes, pipet tips, disposable transfer pipets, a foam microtube holder, a DI water bottle, a sharpie to label, a hot water bath, Non-GMO food, Test food, and a centrifuge. Our materials for the setup of the PCR reactions included: PCR tubes, screwcap tubes, pipet tips, an aerosol barrier, a foam microtube holder, a PCR tube holder, a 2-20 microliter micropipette, an ice bath, a sharpie to label, a plastic bag, a styrofoam cup with ice water, and a black tray with ice water. Finally, the materials for the electrophoresis of the PCR products included: Agarose gel, PCR sample tubes from previous labs, PCR tube holders, running buffers (300-350 ml), loading dye, a PCR molecular weight

Other processes to locate defective genes in the human genome are the Polymerase Chain Reaction (PCR) developed by Kary Mullis and Restriction Fragment Length Polymorphisms (RFLPS), and a technique known as Southern Blotting. The DNA sample can be in creased in amount in a matter of a few hours by using the PCR technique. The DNA is then cut with a restriction enzyme, and the DNA breaks up into restricted fragments. A radioactive probe is added to the fragments that only bonds to certain codons in the DNA. The fragments are subjected to gel electrophoresis. The fragments move through the gel at different rates due to their different lengths. After the electrophoresis is done the DNA on the gel is denatured by heating and the single strands are t ransferred to membrane in a process known as Souther Blotting. The paper is then exposed to photographic film, and the radio active probe exposes itself onto the film which is then processed and studied to find any specific fragment or gene which is the cause of a condition or a disease.

The Blue sample, which was hypothesized to be recombinant, should have produced similar if not identical bands to the known recombinant Green sample, which is not the case in the PCR gel. Instead, the Blue sample shows faint bands more closely resembling the Red sample, known to be non-recombinant. The bands did not show through for the PCR samples which indicates that the PCR preparation was not successful. This is further confirmed when analyzing the positive control which produced no results. The lack of bands in the negative control suggest that failure this portion of the experiment was not due to

Then 5µl of DNA samples were transferred into an individual Eppendorf tube containing the master mix solution. Once completed all 15 tubes were immediately transferred to a thermal cycler.

Polymerase chain reaction (PCR) is a technique used in making multiple copies of DNA segment. The director of the Australian Museum mention that Polymerase Chain Reaction (PCR) was a very important step in producing adequate amounts of Tasmanian Tiger DNA in order to proceed the research and complete the cloning project (Research of the Australian Thylacine N.D). Furthermore Geneticists working for the Australian Museum had successfully replicated Thylacine DNA by using this process.

The first is to denature dsDNA through heating to ~96 °C. This separates the two strands of DNA. The exact temperature to be used can be calculated with Tm = 4oC x (no. of G & C) + 2oC x (no. of A & T). Tm is the melting point of the strands and to supply the number of G, C, A, & T ‘s the primer is used.