Nucleic Acid Measurements

20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.

Purpose The main purpose of this experiment was to test and observe how DNA molecules are being tested or separated. Introduction The final goal of this lab was to successfully measure the size of different samples of DNA placing each samples into a well in agarose gel and running a current through a charged chamber.

The second-grade standard 2-LS4-1 of the New Generation Science Standards” is found under the discipline of Biological Evolution: Unity and Diversity. The standard officially reads, “Make observations of plants and animals to compare the diversity of life in different habitats” (2-LS4 Biological Evolution: Unity and Diversity, n.d.). Putting it more simply however, the standards asks students to look with their eyes and observe, measure, and collect data. Students are meant to look at plants and animals, of a particular area, and identify their characteristics; then students compare the findings to animals and plants of another area. For the purposes of this assignment, the geographic region in question is that of Lake Michigan

Extracted RNA (1 μg) was reverse transcribed using high-capacity cDNA reverse transcription kit, supplied by Applied Biosystems, USA according to the manufacturer's instructions. The cDNA product was diluted in 200 μL Nuclease-free H2O.

Isolation of DNA is mainly done by adding different other digestive enzyme which reacts with constituents in the sample encountered at the crime scene, other than DNA. The different enzymes are proteases which breakdowns amino acids and proteins RNAase which digest RNA particles thus leaving only DNA in the test-tube. Ice cold ethanol is added to this homogenate to precipitate DNA. The DNA can be isolated slowly stirring a glass rod which is known as spooling.

Gel electrophoresis is a laboratory method that allows for the separation of nucleic acids (DNA or RNA) and proteins based on their size. It utilizes a porous gel matrix through which proteins or nucleic acids migrate. Both nucleic acids and proteins can innately and synthetically possess a net-negative electrical charge, a property that is leveraged to facilitate the migration of the desired molecule through the medium. Gel electrophoresis can be conducted in either a horizontal or vertical orientation. Horizontal gels are typically composed of an agarose matrix, while vertical gels are generally composed of an acrylamide matrix.

The process of PCR has 3 steps that repeat in cycles shown in image 1. The first one is the separation of the two strands of the DNA molecule. This happens by the initial material being heated to 95 °C. Every DNA

Once my DNA has been sent to a lab for analysis, a sequencing method can be used to tell me what my DNA sequence is. Because there is the potential for the sample that I sent to not have a great deal of DNA available, polymerase chain reaction may be used to amplify the DNA to get a better sample to analyze. In polymerase chain reaction, they will take the small sample of DNA that I have provided along with two oligonucleotide primers, Taq polymerase, and four deoxynucleotide triphosphate and amplify the sample of DNA. In the first step, the DNA is heated to 95 degrees Celsius in order for the two strands of DNA to separate from each other. After the solution has cooled down to 54 degrees Celsius, short DNA primers hybridize to the DNA. Finally, after raising the temperature to 72 degrees

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5 Physical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 4
 5.1 Landforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 4
 5.2 Climate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 4
 5.3 Flora & Fauna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 4
 5.4 Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The DNA sample to be sequenced is combined with a primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled,

PCR requires DNA polymerase. The specific name for the DNA polymerase that is being used in PCR is Taq polymerase. Taq polymerase is Thermus Aquaticus. The only way Taq polymerase can be used is if it is given a primer. In PCR, the researcher or student is required to choose the primer for the region of DNA that will be replicated. PCR primers are short, they are only about 20 nucleotides long. Two primers are used to make a sequence for the specific DNA region that is being replicated. They

The iScript reverse transcription supermix kit for RT-qPCR (Bio-Rad) was used for cDNA synthesis. Master mix was prepared on ice by adding 4 µl 5x RT supermix (iScript RNase H+ MMLV reverse transcriptase, RNase inhibitor, dNTPs, oligo(dT), random primers, buffer, MgCl2, and stabilizers) and 8 µl nuclease-free water for a single reaction. RNA sample was vortexed and 8 µl was added to each PCR tube. Master mix was thoroughly mixed and 16 µl of master mix was added to each PCR tube. PCR tubes containing 20 µl reaction mix were placed in a thermal cycler and reaction protocol was set as follows: Priming for 5 minutes at 25oC, reverse transcription for 30 minutes at 42oC, reverse transcriptase for 5 minutes at 85oC, and hold for a ∞ time at 4oC.

PCR (Polymerase Chain Reaction) is a laboratory technique that takes specific fragments of DNA and amplifies them using primers and a polymerase that can withstand high temperatures. The materials needed to complete this experiment include the DNA fragments that are to be amplified, two primers (one to attach to the top strand of the DNA fragment of interest and one to attach to the bottom strand on the other end), Taq DNA polymerase, dNTP mix, MgCl2, PCR buffer and the PCR machine. Primers are necessary for the Taq DNA polymerase to attach to in order to begin copying because primers can only attach to an existing piece of DNA. Taq DNA polymerase is necessary for PCR rather than DNA polymerase found in our bodies because when the DNA fragments of interest are heated in order to denature them and separate the two complementary strands, DNA polymerase also denatures. Taq DNA polymerase is taken from a strain of bacteria (Thermus aquaticus) that is taken from the hot springs of Yellowstone National Park, and it can withstand boiling temperatures which makes it effective for PCR. dNTP mix is a mixture of nucletotides that the polymerase uses to create the new

The mixed PCR product was loaded into the gel, and the electrophoresing began in order to separate genomic DNA. The second part of the experiment PCR reaction was DNA sequencing; which was processed by the DNA Learning Center in Cold Spring Harbor, NY. The purpose of sequencing DNA was to determine the exact sequence of nucleotide in a given piece of DNA. The third portion was processed by the lab assistance. The purpose of the third method DNA repair was sequencing to test the effect of UV-light on two different strains of yeast for identifying cell with a mutation required for NER. The fourth process of PCR is DNA purification and RFLP. In DNA purification, QIAquick PCR Purification Kit was used to purify amplicons from other contaminants following the steps in material and methods. For RFLP analysis, the purified amplicons were cut using JOSH04 and JOSH05 PCR reaction with two different restriction enzymes. MsaI and RsaI were used to cut mtDNA control regions from the purified DNA. The fifth method of PCR was running the RFPL sample on gel electrophoresis. Most people within a population have many single nucleotide differences within their genomes. These differences are often

This made use of household detergent as a surfactant to allow the DNA out of the cell and its contents. This method is cheaper to run, not involving costlier laboratory chemicals like Proteinase K. (Ciulla, Sklar and Hauser, 1988, page 488)