3.6.5. Southern hybridization Southern blot hybridization analysis was carried out using standard procedures (Sambrook et al. 2001). Approximately 20 µg of total genomic DNA from transgenic plants and untransformed control plant was digested with EcoRI‚ and then separated by electrophoresis on 0.8% agarose gel. Digestion profile was checked with 1µg DNA‚ with single cutter enzymes EcoRI (10U) in T-DNA region. For southern experiment‚ 20µg DNA was digested overnight. Restricted fragments were separated
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Protein Isolation: Protein isolation for a western blot uses detergents and mechanical force to separate seeded cells from their container. Eukaryotic cells are attached to the surface of a flask by cadherins. In the past‚ we’ve separated the cells from the flask by breaking these bonds with a protease‚ but in order to keep the proteins intact‚ a different method needs to be used to extract the proteins. In protein extraction for a western blot‚ we use detergents to lyse the membrane and free the
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Southern Blotting Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify‚ DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example‚ Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity
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Beta-Galactosidase and Western Blot 0 1. EMG 9 and EMG 26 contain strain _lac-_(I- Z+ Y+) and strain _lac -_ (I+ Z- Y-)respectively.Three genes huddled together on the chromosome are required for two strains of _E.coli_ to utilize lactose.Consisting of three genes‚ namely‚ _lacZ_‚ _lacY_ and _lacA_‚ the _lac_ operon orderly handles these genes to code specific enzymes necessary for the metabolism of lactose. The genes _lacZ_‚ _lacY_ and _lacI_ would code for beta-galactosidase‚ galactosidase permease
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Use of SDS-PAGE/Western Blot Techniques as Diagnostic Tools for Equine Medical Conditions SDS-PAGE Overview Electrophoresis is commonly used to separate proteins using an applied electrical field. SDS-PAGE separates proteins by molecular weight‚ using sodium dodecyl sulfate (SDS; also known as lauryl sulfate) to denature them and a discontinuous polyacrylamide gel as a sieving matrix. The net charges of folded proteins are not dependent upon molecular weight. Rather‚ charge is determined by
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Natalia Rey 080927440 BI338 Lab 1 Measuring the Success of Pisum sativum leaf cell fractionation: SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis Introduction In order to evaluate the success of cellular fractionation‚ there are various methods that can be implemented that target specific proteins and/or receptors specific to particular organelles. SDS-PAGE (sodium dedocyl sulfate- polyachrylamide gel electrophoresis) is a technique used to separate proteins based on
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Southern Gothicism and Dark Romanticism Since the Southern Gothic movement in literature was originated in late 18th and early 19th century‚ it shares some similarities with Dark Romanticism such as death‚ decay‚ and toxic relationships; however‚ Southern Gothicism is a genre of literature that has a very distinctive style of writing. In other words‚ while some of the main characteristics of Dark Romanticism have human imperfections along with the horrific symbols and themes‚ the Southern Gothic
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The Southern Company is a utility holding company whose principal subsidiaries provide power in Alabama‚ Georgia‚ Florida‚ and Mississippi. With the gradual deregulation of the utilities industry in the 1990s‚ Southern Company expanded its holdings into New England‚ New York‚ the Midwest‚ and California‚ making it the largest power producer in the country. In addition‚ Southern now owns power generating operations in Europe‚ Hong Kong‚ the Philippines‚ and South America. The early 1980s saw a
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problems and threats which have existed or currently exist in Southern California and how these problems shape the way we live today and in the imminent future as well. Although Davis did not really provide us with any remedies for the problems facing Southern California‚ this book made it very clear to the readers that problems do still exist‚ although at times they may sound subtle in nature. Of the numerous problems which do exist in Southern California‚ I will discuss only a handful of the problems
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western blot analysis to detect proteins from Non-fat powdered milk. The experiment had to be run two times before the expected outcome was reached. However‚ after the second attempt‚ it was successful. The gel ran well‚ and the proteins from the gel were successfully transferred to the membrane provided in the kit. Introduction The name western blot was given to the technique by W. Neal Burnette and is a pun on the name Southern blot. Southern blot was a name given by Edwin Southern to a
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