Team:Illinois/Protocols

From 2009.igem.org

(Difference between revisions)
(→sRNA Characterization)
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''sRNA Expression Plasmid''
''sRNA Expression Plasmid''
-
1. 50μL PCR reaction
+
1. 50μL PCR reaction (plasmid backbone)
*1μL (10-50 ng) pJU-334 template
*1μL (10-50 ng) pJU-334 template
*0.4μL oligonucleotide pLlacOB
*0.4μL oligonucleotide pLlacOB
Line 39: Line 39:
3. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 30μL water.
3. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 30μL water.
-
4. 30μL digestion reaction
+
4. 30μL digestion reaction (plasmid backbone)
*25μL eluted DNA
*25μL eluted DNA
*3μL 10x Tango buffer
*3μL 10x Tango buffer
Line 46: Line 46:
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.
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.
== '''Recipes''' ==
== '''Recipes''' ==

Revision as of 17:18, 7 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)]

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 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.

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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