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20-September-2008 09:29:09 - Cloning vector The pGEX-3x plasmid is a popular cloning vector. The pGEX-3x plasmid is a popular cloning vector. A cloning vector is a small DNA vehicle into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages such as phage λ are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes BACs and yeast artificial chromosomes YACs. Contents 1 Common Features 2 Screening: example of the blue/white selection 3 See also 4 References Common Features Most commercial cloning vectors have key features that have made their use in molecular biology so widespread. In the case of expression vectors, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism eg. E. coli. Control of expression can be very important; it is usually desirable to insert the target DNA into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters, lac promoters bla promoter and cauliflower mosaic virus's 35s promoter for plant vectors. To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites engineered out of them and a synthetic multiple cloning site MCS inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as an antibiotic resistance eg. beta-lactamase see figure is often carried by the vector to allow the selection of positively transformed cells see Screening below. All plasmids must carry a functional origin of replication ORI; not shown in figure. Some other possible features present in cloning vectors are: vir genes for plant transformation, intergrase sites for chromosomal insertion, lacZα fragment for α complementation and blue-white selection, and/or reporter genes in frame with and flanking the MCS to facilitate the production of recombinant proteins eg. fused to the Green fluorescent protein GFP or to the glutathione S-transferase see figure. Screening: example of the blue/white selection Main article: Blue white screen Many general purpose vectors such as pUC19 usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for E. coli β-galactosidase, whose integrity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal 5 bromo-4-chloro-3-indolyl-beta-d-galactoside into an insoluble, blue product 5,5'-dibromo-4,4'-dichloro indigo. Cloning a fragment of DNA within the vector-based gene encoding the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA. See also Vector DNA Vector molecular biology Expression vector IMAGE cDNA clones References B. R. Glick and J. J. Pasternak 2005. Molecular Biotechnology Principles and Applications of Recombinant DNA. 3rd ed. ASM Press Washington, D. C. April 2007 Retrieved from http://en..org/wiki/Cloning_vector Categories: Molecular biologyHidden categories: Articles lacking sources from April 2007 | All articles lacking sources Views Article Discussion this page History Personal tools Log in / create account Navigation Main page Contents Featured content Current events Random article Search Go Search Interaction Community portal Recent changes Contact Donate to Help Toolbox What links here Related changes Upload file Special pages Printable version Permanent link Cite this page Languages Español Polski Tiếng Việt This page was last modified on 31 May 2008, at 15:58
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So many of us do not eat a balanced diet, get enough sleep, have too much stress, or are impacted with toxins and pollutants. Drinking 2 ounces of MonaVie twice a day will help your body detoxify as well as build your immune system. Its the smartest thing you can do for yourself, so start today. Buying MonaVie through our company guarantees you support 7 days a week and, if you would like to share MonaVie with your family and friends we will guide you from start to finish.
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