2010 Bio 311 Lab D Manual (part 1 + 2)

2010 Bio 311 Lab D Manual (part 1 + 2) - D1. RNA...

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103 D1. RNA Interference in C. elegans Lab Overview: You will perform RNA interference (RNAi) in Caenorhabditis elegans (a nematode worm) using an RNAi “feeding” technique. In this technique, DE3 E .coli , that can make T7 RNA polymerase, will be used to produce. First you will need to spread E. coli bacteria that have been transformed with the RNAi plasmids onto Nematode Agar plates. One of the RNAi plasmids produces double-stranded Unc-22 RNA upon induction with IPTG. Another of the RNAi plasmids produces double-stranded lsy-2 RNA upon induction with IPTG. Also one of the plasmids is the empty RNAi vector. This week you will transfer C. elegans onto the plates where they will consume the E.coli and uptake the dsRNA. You also need to mark your chemotaxis plates for next week’s analysis. Background The soil nematode, Caenorhabditis elegans ( C. elegans ), is a well-studied laboratory model organism that offers a great potential for genetic analysis, partly because of its rapid (3- day) life cycle, small size (1.5-mm-long adult), and ease of laboratory cultivation. Its name is derived from latin: Caeno meaning “recent”, rhabditis meaning “rod-like”, and elegans meaning “nice or beautiful”. Thousands of these animals can be grown on a single Petri dish seeded with a lawn of Escherichia coli as the food source. C. elegans comes in two sexes: a male and a hermaphrodite. The males represent less than one-tenth of a percent in a typical population of C. elegans , and are difficult to find on a dish. The hermaphrodite can reproduce by self-fertilization. Therefore only a single hermaphrodite worm is needed to start or maintain a population. A single worm can produce 300 - 350 offspring in its short lifespan. The entire C. elegans genome was sequenced in 1998, making the C. elegans the first multi-cellular organism to have its genome fully sequenced. The genome sequence data have emphasized the considerable conservation of biological mechanisms and processes across the animal kingdom as a model system has contributed significantly to our biological understanding of many organisms including humans. By the 1990’s, researchers surprisingly discovered that both sense and anti-sense RNA transcripts and double stranded RNAs led to apparent gene inactivation in a number of organisms. The inhibitory effect of double-stranded RNA was studied in C. elegans and * ± Read "the Lab Overview” of Lab D1 and 2. ± State lab Goal(s) . ± Outline or make a flow chart of the methods.
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104 plants. The RNA-mediated interference mechanism has now been solved and is called RNAi. Once taken up by a cell, double-stranded RNA is cleaved into small interfering RNA (siRNA) molecules (~23 bp long) by an enzyme called Dicer. These subunits then bind to a complex known as RISC (RNA-induced silencing complex), where the dsRNA is unwound and then bound to complementary cellular mRNA molecules. The cellular mRNA molecules are then systematically degraded by another enzyme called Slicer. The RNAi mechanism
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This note was uploaded on 03/28/2011 for the course BIO 311 taught by Professor Staff during the Fall '08 term at SUNY Stony Brook.

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2010 Bio 311 Lab D Manual (part 1 + 2) - D1. RNA...

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