Introduction: We were able to examine the DNA or RNA by placing the contents in an agarose gel buffers utilizing an electric charge. The process uses restriction enzymes which cut DNA at restriction sites. The process pushes negatively charged particles to the positive pole. The DNA fragments are used in a gel slab; then it is placed among a buffer solution which utilizes the electricity. The fragments move through the buffer, and it is largely determined by size in which moves quicker. The remains of the same size will join as a single strand which is seen after staining.
Materials and Methods: In order to complete this experiment we utilized micro test tubes labeled appropriately for each enzyme stock solution (Lambda DNA and Restriction Buffer). We utilized colored test tubes for labeling as follows:
• Yellow tube labeled ‘L’ for Lambda DNA
• Violet tube labeled ‘P’ for Pstl lambda digest
• Green tube labeled ‘E’ for EcoRl digest
• Orange tube labeled ‘H’ for Hindlll digest
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Then we pulse-spinned the contents for allowing flow to the bottom. Following, we utilized a floating rack to allow incubation for thirty minutes. After incubation, we proceeded to load 2 micro liters of loading dye into each individual test tube. Following, we allowed the samples to rest on ice. Since we utilized agarose gel, at this point we obtained the gel and filled the chamber with the buffer. The buffer was loaded properly, so we had it properly cover the gel just enough, and it covered both negative and positive poles at the proper height. Next, we loaded ten micro liters in each respective well in the gel. Following, we covered the chamber and connected the leads. Then allowed current to flow for 43 minutes is how long it took our group. The handout recommended approx. 30
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
Upon completion of the experiment we were able to examine the DNA. First, the electrophorese
The given DNA ladder sample and each individual ligation samples were run on 40ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed track dye was used after it was diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed.
.2 ml enzyme 1 ml of 0.0003M DPIP was added to each tube, noting the time the addition was made. A dropper full of cooking oil was added to cover the surface of the solution. The time taken for the blue colour to disappear or become very faint, was recorded. This procedure was repeated for tubes 2 and 3
I would suggest to students performing the nitration to make sure their benzoic acid product is very fine and broken up before reacting it, as it has a tendency to clump together when it dries and thus proves very difficult to react in solution. I would also suggest keeping a very close eye on the temperature when adding the sulfuric/nitric acid mixture dropwise, as the reaction has a tendency to spike in temperature
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
Planaria are one of many free-living flat worms that can be found in marine, aquatic, and terrestrial environments. Certain characteristics of planaria worms include an acoelomate body, a gut with no anus, lack of a blood vascular system, and a simple nervous system. The main reason as to why planaria are subjected to many studies is because of their unique ability to regenerate. Regeneration is the ability to re-grow lost body parts that may have been cut off. This is possible because the organism has the ability to form a blastema, which is an accumulation of undifferentiated cells, at the site of the wound. Regeneration is capable of occurring at various degrees throughout the animal kingdom. This unique process would never be able to be seen in human beings. Humans and other mammals
In biology class, we were learning about enzymes. Enzymes are proteins that help catalyze chemical reactions in our bodies. In the lab, we were testing the relationship between the enzyme catalase and the rate of a chemical reaction. We predicted that if there was a higher percentage of enzyme concentration, then the rate of chemical reaction would increase or it would take less time. We placed 1 ml of hydrogen peroxide into four depressions. Underneath the first depression, we place 1 ml of 100% catalase and make 50% dilution with 0.5 ml of water. We take 50% of that solution and dilute with 0.5 ml of water and we repeat it two more times. there were four depressions filled with catalase: 100%, 50%, 25% , 12.5 % with the last three diluted
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
...l electrophoresis (SCGE) also known as comet assay has become one of the standard methods for assessing DNA damage, with applications ranging from testing genotoxicity, human bio-monitoring and molecular epidemiology to its use in fundamental research in DNA damage and repair (Collins, 2004). The comet assay is a simple method for detecting DNA strand breaks within cells in eukaryotes. The procedure of comet assay includes Embedding the cells in agarose in a microscope slide, followed by lysing of cells with detergent and high salts to form nucleotides containing supercoiled loops of DNA linked to the nuclear matrix, and then undergoing Electrophoresis at high pH, which results in formation of structures resembling as comets, observed by fluorescence microscopy. The intensity of the comet tail relative to the head reflects the number of DNA breaks (Collins, 2004).
In this lab, it was determined how the rate of an enzyme-catalyzed reaction is affected by physical factors such as enzyme concentration, temperature, and substrate concentration affect. The question of what factors influence enzyme activity can be answered by the results of peroxidase activity and its relation to temperature and whether or not hydroxylamine causes a reaction change with enzyme activity. An enzyme is a protein produced by a living organism that serves as a biological catalyst. A catalyst is a substance that speeds up the rate of a chemical reaction and does so by lowering the activation energy of a reaction. With that energy reactants are brought together so that products can be formed.
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
Restriction enzyme will cleave a piece of DNA into a series of fragments. The number and sizes of the fragments depend on the number and location of restriction sites for that enzyme in the given DNA. A specific combination of bases will occur at random only once every few hundred bases, while a specific sequence of 6 will occur randomly only once every few thousand
The restriction enzymes SmaI cuts DNA vertically. This results in two DNA fragments with blunt ends. Next, the gene is spliced into a vect... ... middle of paper ... ... le by stopping illness but this process has also been vandalised for many uses which are not necessary.