Team:Illinois/Protocols

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Contents

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)]

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.

Procedure:

sRNA Expression Plasmid

1. 50μL PCR reaction

    • 1μL (10-50 ng) pJU-334 template
    • 0.4μL oligonucleotide pLlacOB
    • 0.4μL oligonucleotide JVO-2164
    • 10μL Phusion buffer
    • 1μL dNTP mix
    • 0.3μL DNA polymerase

Run for 30s @ 98°C, then 30 cycles of the following:10s @ 98°C, 30s @ 58°C, 2 min 20s @ 72°C. Incubate for 5 min @ 72°C, then analyze 5μL of the reaction in 0.8% agarose gel, looking for a ~3.1kbp fragment. 2. Mix in 1.5μL of DpnI with remaining 45μL of reaction, incubate for 3 hr at 37°C. 3. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 30μL water. 4. 30μL digestion reaction

    • 25μL eluted DNA
    • 3μL 10x Tango buffer
    • 2μL XbaI

Digest for 6 hr at 37°C, then add 1μL shrimp alkaline phosphatase (SAP) and incubate for 1 hr at 37°C.

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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