In molecular cell biology, restriction enzyme is used in various ways to understand the structure of a DNA molecule and how each of fragment of the sequence works. For this experiment, many researchers use several techniques and come up with logic questions that have various results to them. Such as in this study, researchers are analyzing the electroporation of BamHI or EcoRI together with pyr5-6 plasmids cuts with the same enzyme, which stimulates the efficiency of transformation in Dictyostelium discoideum (Kuspa and Loomis, 1992). This content shows the comparison between two different enzymes and how each fragments is affected with the source of pyr5-6 plasmid. Other studies use the three enzymes BamHI, EcoRI and HamIII together in numerous ways to get valuable results. Another technique is by constructing a restriction map, which is used in the lab. …show more content…
In this experiment, we perform a gel electrophoresis on the DNA. In this process, the enzymes were run through the gel electrophoresis to determine their relative sizes for each of them. The results of the certain DNA fragments are used in the final step, which is to construct a map of the DNA molecule. If we use different enzymes to cut DNA, then not every restriction site will be cut by all of the enzymes. The objective of this lab is to perform restriction enzyme of digesting plasmid DNA and constructing a map of plasmid from the results made from the experiment. Using this technique we understand what a DNA restriction enzyme is and how it works. In this process, the enzymes were run through the gel electrophoresis to determine their relative sizes for each of them. By following the experiment, we determined the positions of the restriction
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
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.
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
One of the most primitive actions known is the consumption of lactose, (milk), from the mother after birth. Mammals have an innate predisposition towards this consumption, as it is their main source of energy. Most mammals lose the ability to digest lactose shortly after their birth. The ability to digest lactose is determined by the presence of an enzyme called lactase, which is found in the lining of the small intestine. An enzyme is a small molecule or group of molecules that act as a catalyst (catalyst being defined as a molecule that binds to the original reactant and lowers the amount of energy needed to break apart the original molecule to obtain energy) in breaking apart the lactose molecule. In mammals, the lactase enzyme is present
The aim of this experiment was to isolate cDNA molecule CIH-1 (Colletotrichum lindemuthianum CIH1 gene) that is contained in vector pBK-CMV and transfer it into cloning vector pUC19. This was attempted by conducting a restriction digest of vectors pUC19 and pBk-CMV containing CIH-1, using restriction endonucleases Xba1 and EcoR1 and the characterization of recombinant plasmids.
As the solution pH can influence the stability of NaClO-NH3 blend and the elimination of SO2, NOx, the impact of the pH of NaClO-NH3 blend solution on the instantaneous removal as well as the duration time was investigated, and the final pH after reaction was also detected and shown in Fig. 5. It can be seen that the variation of solution pH has a negligible effect on the desulfurization, but the elevated pH has a great promotion on the NOx removal, the efficiencies are significantly increased from 36% to 99% for NO2 in the pH range of 5–12 and from 19% to 65% for NO when the pH is between 5 and 10, after where, both of them are constant. Hence, the optimal pH of the NaClO-NH3 solution for the
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
The study of nucleic acids has now become a fruitful and dynamic scientific enterprise. Nucleic acids are of unique importance in biological systems. Genes are made up of deoxyribonucleic acid or DNA, and each gene is a linear segment, or polymer, of a long DNA molecule. A DNA polymer, or DNA oligonucleotide, contains a linear arrangement of subunits called nucleotides. There are four types of nucleotides. Each nucleotide has three components; a phosphate group, a sugar and a base that contains nitrogen within its structure. The sugar moiety in DNA oligonucleotides is always dexoyribose, and there are four alternative bases: adenine (A), thymine (T), guanine (G), and cytosine (C). The phosphate groups and the deoxyribose sugars form the backbone of each DNA stand. The bases are joined to the deoxyribose sugar and stick out to the side. Both oligomers, DNA and RNA, consist of 5’->3’ phosphodiester-linked nucleotide units that are composed of a 2’-deoxy-D-ribose (DNA) or D-ribose (RNA) in their furanose forms and a heteroaromatic nucleobase (A, T, G, and C; A, U, G, C), and the resulting oligonucleotide chain is composed of a polar, negatively charged sugar-phosphate backbone and an array of hydrophobic nucleobases. The amphiphilic nature of these polymers dictates the assembly and maintenance of secondary and tertiary structures the oligonucleotides can form. In the DNA duplex structure, genetic information is stored as a linear nucleotide code. This code can be accessed and replicated. RNA, or ribonucleic acid, is another structurally related essential biopolymer. RNA differs from DNA in having the sugar ribose in place of the deoxyribos...
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...
Materials used in the experiment included 5-7 g of the potato tissue, 50ml of 2.0M phosphate buffer coffee filter and guaiacol dye.
In this experiment the enzyme peroxidase and the substrate hydrogen peroxide were not mixed initially, instead they were both placed in separate tubes and were incubated at a specific temperature, to prevent hydrogen peroxide from undergoing any reaction with peroxidase until they both acquire the required temperature.
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.
Secondly the gene has to be cut from its DNA chain. Controlling this process are many restriction endonucleases (restriction enzymes). Each of these enzymes cut DNA at a different base sequence called a recognition sequence. The recognition sequence is 6 base pairs long. The restriction enzymes PstI cuts DNA horizontally and vertically to produce sticky ends.