Dna And Multiple Cloning Sites Essay - 1407 Words

Introduction In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the ÃŽ ²-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016). The addition of 5-bromo-4-chloro-3-indoyl-ÃŽ ²-thiogalactoside (X-gal) in the agar mix acts as an artifical substrate for the enzyme so it produces blue E.coli colonies when hydrolised by the ÃŽ ²-galactosidase meaning those specific colonies do not contain the plasmid with the CIH-1 insert (non recombinant E.coli) (Coventry University 2016). Isopropyl-ÃŽ ²-D-thiogalactoside (IPTG) an artifical transcription inducer of the lacZ gene is also added into the agar mix, binding to the repressor gene and inactivating it. Therefore when the CIH-1 gene is incorporated within the plasmid MCS and inserted into the E.coliShow MoreRelatedPolymerase Chain Reaction Essay1003 Words   |  5 PagesOne may view cloning as copying a living thing and producing multiple copies. People may think of cloning rabbits, sheep or humans. In the field of molecular biology, however cloning is viewed at a genetic molecular level, where a piece of DNA is copied on a large-scale by genetically copying tens to hu ndreds of thousands of identical DNA fragments. Researchers are developing new methods of cloning by using polymerase chain reaction (PCR). PCR was introduced in the 1980s and in recent years KaryRead MoreEscherichia Coli : Is A Well Studied Gram Negative Bacteria916 Words   |  4 Pageslevel of DNA supercoiling, an important property of DNA and chromatin (Gilbert and Allan, 2014). The supercoiling level is modified by a reaction that consists in the transient breakage of the DNA phosphodiester bonds and the movement of strands across the transient breaks (Tse-Dinh and Wang, 1986). In prokaryotes, these enzymes can be classified into two major groups, according to the mechanism of action. While the topoisomerase II (DNA gyrase) generates negative supercoils into relaxed DNA and relaxRead MoreDna Lab Report1345 Words   |  6 Pages The genomic DNA sample concentration of 28.5 ng/ÃŽ ¼L and its A260/A280 ratio of 1.85 indicates that the DNA was relatively pure since a 260/280 ratio of ~1.8 is generally accepted as pure for DNA (Cox, Doudna O’Donnell, 2015). The ratio of white colonies to blue colonies observed on the blue-white screen plate was 31:37, which shows that there was a greater number of E. coli DH5-ÃŽ ± that did not contain the plasmid with the foreign gene insert. Therefore, out of all of the E. coli DH5-ÃŽ ± cells thatRead MoreVibrio Fischeri Is A Bacterium That Served As The Model Organism802 Words   |  4 Pagesexperiment, Vibrio fischeri chromosomal DNA was isolated by lysing the bacterial membranes and removing the proteins and lipids. Addition of a lysozyme solution breaks down the peptidoglycan layer in Gram-negative bacteria and proteinase K/SDS degrades the proteins and disrupt the membranes. Liquid-liquid extraction using Tris-buffered phenol permits separation of proteins and lipi ds from the DNA into the organic layer. It is also essential that the isolated DNA is pure and free of proteins, sugarsRead MoreA Segment Of The Will Die Slowly Gene From Drosophila Melanogaster1688 Words   |  7 PagesTitle: â€Å"PCR amplification, TA cloning, sequencing and sequence analysis of a segment of the will die slowly gene from Drosophila melanogaster.† Abstact: A seemingly novel gene sequence has been discovered in fruit flies from Nicholas Harden’s lab at Simon Fraser University using random PCR primers. Attempts at purifying and characterizing this gene have been elusive. Here, we show methods for isolating, amplifying, and purifying the gene of interest for analysis. Using polymerase chain reactionRead MoreEssay On Mutation1657 Words   |  7 Pagesframeshift mutations. How are these mutations the same and how are they different? Silent, missense, and nonsense mutations all occur when base substitutions happen (text p.206). A base substitution is when the wrong nucleotide is incorporated during DNA synthesis, this is the most common mutation (lecture). In a silent mutation a base substitution occurs, incorporating an incorrect nucleotide, the result is a codon that codes for an amino acid that is the same as the wild type (text p. 206). In a missenseRead MoreVectors Used in Biotechnology2739 Words   |  11 Pagesclosed circula r, double-stranded, extrachromosomal DNA molecules which occur naturally in bacteria, yeast, and some higher eukaryotic cells, and exist in a parasitic or symbiotic relationship with their host cell (Lodish et al., 2000) The main application of plasmids is as cloning vectors in gene cloning. In gene cloning, a fragment of DNA, containing the gene to be cloned is inserted into a circular molecule called the â€Å"vector† to produce recombinant DNA molecule. Plasmids are one of the most commonlyRead MoreGene Regulation For Safety Assessment946 Words   |  4 Pagesapplied to all procedures, and universal precautions and lab safety protocols are followed. Transformation Methods: 1. The Isolation of Gene of Interest from Pseudomonas putida 2. The Cloning Process 3. Transformation of bacteria Quality Control: 1. Selection and screening of Recombinant cells 2. Purification of DNA 3. Purification and Storage of Bacteria. 1. The Isolation of Gene of Interest from Pseudomonas putida: The isolation of P.putida strain was carried out by using P-2 media. This mediaRead MoreStem Cell Research1167 Words   |  5 Pages. Stem cell is a cell that has the ability to change into several or multiple types of cells or into any cell type in the body. There are two main types of stem cells, adult stem cells and embryonic stem cells. Embryonic stem cells are found in cells that have only undergone the first few dozen cycles of growth and division following the fusion of egg and sperm. These cells are pluripotent and can be maintained so that they grow and divide indefinitely without differentiating. Likewise, theyRead MoreDescription And Function Of Mutagenesis1345 Words   |  6 PagesSite-directed mutagenesis is a method used to construct amino acid changes in a protein in order to test the function of a specific amino acid. It is the way to study protein structure and function change the amino acid sequence of a protein by altering the DNA sequence of cloned gene4. A particular amino acid is very important in catalytic activity, ligand binding, protein folding or other function. Amino acid residue significance is tested by making conservative substitutions or by changing the