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Team:Illinois/Protocols
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This protocol is used to characterize an E. coli small RNA and its ability to downregulate a reporter gene (GFP). The small RNA is cloned out of the E. coli chromosome and inserted into a high-copy number plasmid (pJU-334). The sRNA's target sequence is also cloned out of the E. coli chromosome and inserted into a low-copy number plasmid (pXG-10). Both plasmids are then transformed into E. coli Top10F' cells. Successful sRNA repression is indicated by a decreased level of fluorescence in the cells. | This protocol is used to characterize an E. coli small RNA and its ability to downregulate a reporter gene (GFP). The small RNA is cloned out of the E. coli chromosome and inserted into a high-copy number plasmid (pJU-334). The sRNA's target sequence is also cloned out of the E. coli chromosome and inserted into a low-copy number plasmid (pXG-10). Both plasmids are then transformed into E. coli Top10F' cells. Successful sRNA repression is indicated by a decreased level of fluorescence in the cells. | ||
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Revision as of 00:04, 18 July 2009
Protocols
This page describes protocols or includes links to protocols used in our project. Recipes used are also listed in a separate section. Protocols are organized by category. They were collected from Open Wet Ware, our advisors, and labs around the University of Illinois campus.
Standard
- [http://www.biochem.northwestern.edu/morimoto/research/Protocols/I.%20Prokaryotes/A.%20Bacteriology.pdf Bacterial Culture Basics (media/plate preparation, growth, streaking, and storage)]
- [http://www.methodbook.net/dna/agarogel.html Agarose Gel Electrophoresis Protocol]
- [http://www.5prime.com/media/29661/perfectprep%20spin%20mini%20kit%20manual_5prime_1048299_072007.pdf Miniprep Protocol (PerfectPrep Spin Mini Kit)]
- [http://www.promega.com/tbs/tb374/tb374.pdf Miniprep Protocol (PureYield Plasmid Miniprep System)]
sRNA Characterization
Taken from: [http://www.springerlink.com.proxy2.library.uiuc.edu/content/h752r3415261357p/ A Green Fluorescent Protein (GFP)-Based Plasmid System to Study Post-Transcriptional Control of Gene Expression In Vivo]: Johannes H. Urban and Jörg Vogel, 2009
This protocol is used to characterize an E. coli small RNA and its ability to downregulate a reporter gene (GFP). The small RNA is cloned out of the E. coli chromosome and inserted into a high-copy number plasmid (pJU-334). The sRNA's target sequence is also cloned out of the E. coli chromosome and inserted into a low-copy number plasmid (pXG-10). Both plasmids are then transformed into E. coli Top10F' cells. Successful sRNA repression is indicated by a decreased level of fluorescence in the cells.
Questions about our Wiki page? Please email us at illinoisiGEM@gmail.com.
