Team:Illinois/Protocols

From 2009.igem.org

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Revision as of 19:40, 10 July 2009

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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.

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)]

BioBrick information

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. sRNA characterization procedure

Recipes

LB Growth Media
- 1L dH₂0
- 10g NaCl
- 5g yeast extract
- 10g Bacto-tryptone

Agarose Gel
- 50mL 0.5x TBE buffer
- Agarose (To determine number of grams to use, multiply volume (50mL) by percentage of gel. For example, use 1.5g of agarose in a 3% agarose gel.)
- 2.5μL ethidium bromide

Questions about our Wiki page? Please email Dave Korenchan at korench1@illinois.edu.

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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.
-[[[https://2009.igem.org/SRNA_Expression_Plasmid]]]
 [[sRNA characterization procedure]]