Analysis of A. fischeri chDNA restriction digestion by Sal I enzyme after isolation and purification via agarose gel electrophoresis Bio 219-064 (Techniques in Molecular Biology) Group 5: Tung Nguyen‚ Uyen Tran‚ Amber Beckley‚ Danielle Exler Department of Biology‚ Drexel University‚ Philadelphia‚ Pennsylvania 19104 Submitted October 27‚ 2014 ________________________________________________________________________ Abstract Bioluminescence is one of the benefits that a deep sea organism received
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histidine-tagged pbluescript in a forward orientation potentially allows isolation of protein via Affinity Chromatography or Chromatin Immunoprecipitation therefore its role‚ effects and targets in the genome can be established. Resultant Recombinant plasmids in this experiment had multiple inserts leading to inconclusive orientation of the inserts; however this can be tackled by Sanger or Maxam/Gilberts sequencing. Introduction The capacity to segregate and amplify individual genes from an intricate
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Brianna Noriega In order to analyze a dna molecule in a molecular biology lab you must determine the length in nucleotide pairs. Electrophoresis is an extremely useful tool in order to compare the mobility on agarose gels with dna markers of known lengths. Dna is a polymer that is negatively charged due to the sugar phosphates. When dna is on an electric field such as the electrophoresis gel the different lengths of dna migrate at different rates when they move through the porous gel. The ends
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Subcloning of fungal cDNA from pBK-CMW into a plasmid vector pUC19 using fungal gene CIH Introduction A plasmid is a circular‚ double stranded DNA molecule that replicates independently of the chromosome DNA within a cell.pUC19 is one of the most commonly plasmid cloning vector used due to its high copy replication number (approx. 100 copies per cell)‚ ampR (ampicillin resistance gene) andterminal fragment of β -galactosidase (lac Z). It is circular double stranded and it has 2686 base pair
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and may share a common ancestry to the a-chain of human b-hexos-aminidase. Chitobiase is encoded by chb. In this experiment‚ a restriction map for restriction enzymes Eco R1‚ Pst1 and Hind III using Southern hybridization and restriction analysis of pRSG 192. pRSG 192 is a recombinant plasmid derived from the chb gene and pUC 19‚ a 2.7kb engineered plasmid which encodes for ampicillin resistance‚ a portion of the lac operon and a multiple cloning region . The chb gene exists as a 3.6 kb
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1. Introduction Since the discovery of plasmid‚ various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction at pH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl
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Transformation of E. coli with your unknown plasmid Learning Goals: Insert your uncut unknown plasmid into chemically competent DH-5 E.coli cells and use antibiotic resistance to confirm the success of the transformation. You should familiarize yourself with the various methods of transformation and the advantages/disadvantages of each type. You should also understand how heat shock transformation works and how chemically competent cells make this type of transformation possible. For this transformation
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to control DNA through the use of plasmid vectors became an attraction for newer technologies‚ which allowed specific changes in discrete‚ manageable segments of the genome with relatively little effort. Site-directed mutagenesis method was first benefited from recombinant DNA technology in 1970s‚ when isolated genes were exposed to conditions such as chemical agents or nucleotide analogs to localize their mutagenic effects. During this time‚ the use of plasmid vectors for DNA replication greatly
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a strand of DNA. 2. Restriction enzymes are made by bacteria to cut up invading DNA. They target specific base sequences in the DNA and then work to cut out those sequences from the DNA. 3. When a restriction enzyme cuts out a portion of DNA‚ it will sometimes leave a sticky end. If two fragments of DNA are cut by the same restriction enzyme‚ the sticky ends can join together‚ forming a recombinant DNA strand. 4. Cut plasmid and eukaryotic DNA with the same restriction enzyme… Mix fragments
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a genomic library‚ genomic DNA from Vibrio fischeri was first isolated then treated with Sal I restriction enzyme to generate inserts (smaller fragments of DNA). Sal I restriction enzyme was also used to treat the vector plasmid in order to digest the V. fischeri DNA fragments. The inserts and the vector were then ligated together. E.Coli cells were then made competent in order to take up the plasmid DNA by transforming these competent cells with a “ligation mixture to create a population of host
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