This experiment was very successful as a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. Within the gel electrophoresis, it is necessary to run a 1kB ladder in the first well. This ladder contains numerous known lengths of base pairs, and is run next to and unknown product in order to approximate the sizes of unknown fragments simply by comparing the unknown fragments to the coinciding fragments of the known ladder. This ladder gives us the ability to precisely and accurately draw conclusions about the results derived from the gel electrophoresis as it serves as an essential reference point. Because of the known fragments in the ladder, we were able to create a standard curve. Within the standard curve, the distance the fragments traveled was plotted against the length of the known base pairs within the ladder. Once the points were plotted, a line of best fit was constructed and an equation of the line was electronically derived. By plugging in the measured distance of how far the fragments traveled, shown by “x”, into the equation for the line of best fit, the lengths of the base pairs created by the restriction enzymes was able to be calculated.
In order to make the final step of mapping out the restriction sites on an unknown plasmid, it was essential to first map out the single digest maps. The single digest maps were created using both the number of fragments produced...
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...lasmid have the capability to survive, and multiple in number as they expand and reproduce. In addition, restriction enzymes have led to gateway discoveries in the topic of cloning. Essentially, because these restriction enzymes have allowed for the removal of a fragment of DNA and for it to be placed in another location, this idea has led to scientists being able to integrate exogenous DNA into natural plasmids that may ultimately lead to cloning plasmid vectors. These plasmids then have the ability to self-replicate (neb.com). The discoveries made surrounding these restriction enzymes have paved the way for the cloning of DNA. Furthermore, DNA mapping is a practical application stemming from restriction analysis that now allows for scientists to be able to detect insertions and deletions, single nucleotide polymorphisms, and identifying genetic disorders (neb.com)
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
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.
The green fluorescent protein (GFP) gene is a naturally occurring gene from a bioluminescent jellyfish. The gene allows for objects and animals to glow in the dark when activated by the presence of the sugar arabinose in the pGLO plasmid. The GFP gene is often used as a marker for gene expression and genetic transformation. The pGLO plasmid is a genetically engineered plasmid used as a vector in biotechnology to generate genetically modified organisms(GMO). M. Chalfie et. al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things.
Glase, Jon C. A Study of Gene Linkage and Mapping Using Tetrad Analysis in the Fungus
...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.
Many things have impacted both the Science and Medical fields of study. Electrophoresis and DNA Sequencing are two of these things. Together they have simultaneously impacted both of these fields. On one hand, there is Electrophoresis. Electrophoresis is a specific method of separating molecules by their size through the application of an electric field. It causes molecules to migrate at a rate and distance dependent on their size. On the other hand, there is DNA Sequencing. DNA Sequencing is a technique used to determine the exact sequence of bases
High school students across the world hate Gene, the character that they are forced to read about, in a book they didn’t want to read. Gene is generally thought of as the despicable human being that ruined his friend’s life. It is easy to write Gene off as a one dimensional character who is only concerned about himself. This common misconception is proved to be false after a deeper analysis of Gene’s character. Gene’s character is more complex than his exterior actions portray. In reality, Gene’s inner “evil” represents a part of human nature, which most people are unwilling to look at in themselves. Gene’s actions throughout the book should not be written off only because Gene is a terrible person. The motivation for Gene’s actions might not
The purpose of this experiment is to identify an unknown insert DNA by using plasmid DNA as a vector to duplicate the unknown insert DNA. The bacteria will then be transformed by having it take in the plasmid DNA, which will allow us to identify our unknown insert as either the cat gene or the kan gene.
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.
Many other words, both positive and negative, spring to mind when one hears the word "mutation." In a scientific sense, one might think of the random variations that lead to evolution in species. In a sci-fi/ horror flick sense, one might think of a vicious monster that after contact with some radioactive substance became terribly disfigured. But rarely do we associate mutations with ideas pervasive to our culture. Daniel Dennett suggests that memes undergo a certain kind of mutation that is inherent to the creative evolution of culture.
Over the last fifty years or so, scientists have made a great amount of progress in this area, including the development of techniques which allow for the controlled manipulation and replication of specific segments of the human genome. These types of techniques have come to be known as recombinant DNA (rDNA) technology and have allowed scientists to analyze functions of genes which are not necessarily directly expressed at the phenotypic level. This is done by "cutting out" or excising a particular segment of DNA of interest from the genetic material of an individual and inserting it into a bacterial plasmid (a tiny ring of DNA in addition to the normal chromosomal material found within the cells of bacteria).
Then the sequence was loaded into Velvet where it was trimmed to the desired k-mer length for alignment and contig formation. Mitos and MEGA alignment Explorer were also used in order to get the DNA sequence to a
The birth of genetic engineering and recombinant DNA began in Stanford University, in the year 1970 (Hein). Biochemistry and medicine researchers were pursuing separate research pathways, yet these pathways converged to form what is now known as biotechnology (Hein). The biochemistry department was, at the time, focusing on an animal virus, and found a method of slicing DNA so cleanly that it would reform and go on to infect other cells. (Hein) The medical department focused on bacteria and developed a microscopic molecular messenger, that could not only carry a foreign “blueprint”, or message, but could also get the bacteria to read and copy the information. (Hein) One concept is needed to understand what happened at Stanford: how a bacterial “factory” turns “on” or “off”. (Hein) When a cell is dividing or producing a protein, it uses promoters (“on switches”) to start the process and terminators (“off switches”) to stop the process. (Hein) To form proteins, promoters and terminators are used to tell where the protein begins and where it ends. (Hein) In 1972 Herbert Boyer, a biochemist, provided Stanford with a bacterial enzyme called Eco R1. (Hein) This enzyme is used by bacteria to defend themselves against bacteriophages, or bacterial viruses. (Hein) The biochemistry department used this enzyme as a “molecular scalpel”, to cut a monkey virus called SV40. (Hein) What the Stanford researchers observed was that, when they did this, the virus reformed at the cleaved site in a circular manner. It later went on to infect other cells as if nothing had happened. (Hein) This proved that EcoR1 could cut the bonding sites on two different DNA strands, which could be combined using the “sticky ends” at the sites. (Hein). The contribution towards genetic engineering from the biochemistry department was the observations of EcoR1’s cleavage of
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.