Team:Illinois/Protocols

From 2009.igem.org

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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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'''Procedure:'''
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[[Procedure]]
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[[sRNA Expression Plasmid]]
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1. 50μL PCR reaction (plasmid backbone)
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*1μL (10-50 ng) pJU-334 template
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*0.4μL oligonucleotide pLlacOB
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*0.4μL oligonucleotide JVO-2164
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*10μL Phusion buffer
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*1μL dNTP mix
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*0.3μL DNA polymerase
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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.
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2. Mix in 1.5μL of DpnI with remaining 45μL of reaction, incubate for 3 hr at 37°C.
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3. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 30μL water.
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4. 30μL digestion reaction (plasmid backbone)
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*25μL eluted DNA
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*3μL 10x Tango buffer
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*2μL XbaI
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Digest for 6 hr (or overnight) at 37°C, then add 1μL shrimp alkaline phosphatase (SAP) and incubate for 1 hr at 37°C.
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5. Purify the ~2.2kbp fragment in 0.8% agarose gel (there should also be a ~0.9kbp fragment), elute DNA in 25μL water using NucleoSpin Extract II DNA purification kit.
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6. 25μL PCR reaction (sRNA gene of interest)
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*1μL (10-50 ng) chromosomal E. coli K12 template DNA
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*0.2μL of each primer (Note: The sense primer pairs with the sRNA gene starting at the +1 transcriptional start nucleotide and is 5'-phosphorylated for blunt-end ligation.  The antisense primer pairs ~40nt down from the terminator and has an XbaI site and five additional nucleotides.)
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*2.5μL 10x Pfu buffer
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*0.5μL dNTP mix
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*0.4μL Pfu DNA polymerase
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*20.2μL water
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Run for 5 min @ 95°C, then 30 cycles of the following: 45s @ 95°C, 45s @ 56°C, 30s @ 72°C. Incubate for 5 min @ 72°C, then analyze 5μL of the reaction in 3% agarose gel.
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7. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 15μL water.
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8. 10μL digestion reaction (sRNA gene of interest)
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*8μL eluted DNA
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*1μL 10x Tango buffer
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*1μL XbaI
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Digest for 3 hr at 37°C.
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9. Purify the proper fragment in 3% agarose gel, elute DNA in 15μL water using NucleoSpin Extract II DNA purification kit.
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10. 5μL small-scale ligation reaction
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*~12ng digested plasmid backbone
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*~5ng digested sRNA gene
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*0.5μL 10x T4 DNA Ligase Reaction Buffer
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*0.5μL T4 DNA ligase
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Incubate 1 hr at room temperature. Include control ligation where DNA insert is replaced by water.
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11. Transform 2μL of reaction into E. coli Top10F' cells. Expect 50-200 colonies.
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''Target-GFP Fusion Cloning''
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1. Inoculate single colony of E. coli Top10 cells containing pXG-10 plasmid into 4mL LB containing 20μg/mL chloramphenicol, grow overnight at 37°C with agitation.
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2. Dilute culture in 400mL fresh LB medium, incubate overnight.
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3. Isolate pXG-10 plasmid using the NucleoBond PC100 plasmid purification kit. Use double volumes of washing buffer in each step mentioned in the manufacturer's protocol. Resuspend DNA in 80μL water.
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4. 60μL digestion reaction (pXG-10)
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*4μg pXG-10 plasmid
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*40μL NheI
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*20μL BfrBI
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*1x Tango buffer
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Digest for 7 hr (or overnight) at 37°C, then add 20μL shrimp alkaline phosphatase (SAP) and incubate for 3 hr at 37°C..
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5. Load reaction on 1% agarose gel and excise the 4.1kbp band. Purify using NucleoSpin Extract II DNA purification kit and elute in 50μL water.
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6. 25μL PCR reaction (sRNA target sequence)
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*1μL (10-50 ng) chromosomal E. coli K12 template DNA
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*0.2μL of each primer
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*2.5μL 10x Pfu buffer
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*0.5μL dNTP mix
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*0.4μL Pfu DNA polymerase
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*20.2μL water
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Run for 5 min @ 95°C, then 30 cycles of the following: 45s @ 95°C, 45s @ 58°C, 45s @ 72°C. Incubate for 5 min @ 72°C, then analyze 5μL of the reaction in 3% agarose gel.
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7. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 15μL water.
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8. 15μL digestion reaction (sRNA target sequence)
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*12μL eluted DNA
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*7.5U NheI
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*7.5U BfrBI
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*1x Tango buffer
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Digest for 3 hr at 37°C.
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9. Purify the proper fragment in 3% agarose gel, elute DNA in 15μL water using NucleoSpin Extract II DNA purification kit.
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10. 5μL small-scale ligation reaction
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*~12ng digested pXG-10
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*~5ng digested sRNA target sequence
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*0.5μL 10x T4 DNA Ligase Reaction Buffer
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*0.5μL T4 DNA ligase
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Incubate 1 hr at room temperature. Include control ligation where DNA insert is replaced by water.
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11. Transform 2μL of reaction into E. coli Top10 cells. Expect 50-200 colonies.
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== '''Recipes''' ==
== '''Recipes''' ==

Revision as of 19:37, 10 July 2009

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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)]
  • [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.

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


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