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Team:Illinois/Protocols
From 2009.igem.org
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 (plasmid backbone)
- 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 (plasmid backbone)
- 25μL eluted DNA
- 3μL 10x Tango buffer
- 2μL XbaI
Digest for 6 hr (or overnight) at 37°C, then add 1μL shrimp alkaline phosphatase (SAP) and incubate for 1 hr at 37°C.
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.
6. 25μL PCR reaction (sRNA gene of interest)
- 1μL (10-50 ng) chromosomal E. coli K12 template DNA
- 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.)
- 2.5μL 10x Pfu buffer
- 0.5μL dNTP mix
- 0.4μL Pfu DNA polymerase
- 20.2μL water
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, looking for a ~160bp fragment.
7. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 15μL water.
8. 10μL digestion reaction (sRNA gene of interest)
- 8μL eluted DNA
- 1μL 10x Tango buffer
- 1μL XbaI
Digest for 3 hr at 37°C.
9. Purify the ~150bp fragment in 3% agarose gel, elute DNA in 15μL water using NucleoSpin Extract II DNA purification kit.
10. 5μL small-scale ligation reaction
- ~12ng digested plasmid backbone
- ~5ng digested sRNA gene
- 0.5μL 10x T4 DNA Ligase Reaction Buffer
- 0.5μL T4 DNA ligase
Incubate 1 hr at room temperature. Include control ligation where DNA insert is replaced by water.
11. Transform 2μL of reaction into E. coli Top10F' cells. Expect 50-200 colonies.
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.
