SRNA Expression Plasmid

sRNA Characterization abstract
Taken from: 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:
1. 50&mu;L PCR reaction (plasmid backbone) Run for 30s @ 98&deg;C, then 30 cycles of the following:10s @ 98&deg;C, 30s @ 58&deg;C, 2 min 20s @ 72&deg;C. Incubate for 5 min @ 72&deg;C, then analyze 5&mu;L of the reaction in 0.8% agarose gel, looking for a ~3.1kbp fragment.
 * 1&mu;L (10-50 ng) pJU-334 template
 * 0.4&mu;L oligonucleotide pLlacOB
 * 0.4&mu;L oligonucleotide JVO-2164
 * 10&mu;L Phusion buffer
 * 1&mu;L dNTP mix
 * 0.3&mu;L DNA polymerase

2. Mix in 1.5&mu;L of DpnI with remaining 45&mu;L of reaction, incubate for 3 hr at 37&deg;C.

3. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 30&mu;L water.

4. 30&mu;L digestion reaction (plasmid backbone) Digest for 6 hr (or overnight) at 37&deg;C, then add 1&mu;L shrimp alkaline phosphatase (SAP) and incubate for 1 hr at 37&deg;C.
 * 25&mu;L eluted DNA
 * 3&mu;L 10x Tango buffer
 * 2&mu;L XbaI

5. Purify the ~2.2kbp fragment in 0.8% agarose gel (there should also be a ~0.9kbp fragment), elute DNA in 25&mu;L water using NucleoSpin Extract II DNA purification kit.

6. 25&mu;L PCR reaction (sRNA gene of interest) Run for 5 min @ 95&deg;C, then 30 cycles of the following: 45s @ 95&deg;C, 45s @ 56&deg;C, 30s @ 72&deg;C. Incubate for 5 min @ 72&deg;C, then analyze 5&mu;L of the reaction in 3% agarose gel.
 * 1&mu;L (10-50 ng) chromosomal E. coli K12 template DNA
 * 0.2&mu;L 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.)
 * 2.5&mu;L 10x Pfu buffer
 * 0.5&mu;L dNTP mix
 * 0.4&mu;L Pfu DNA polymerase
 * 20.2&mu;L water

7. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 15&mu;L water.

8. 10&mu;L digestion reaction (sRNA gene of interest) Digest for 3 hr at 37&deg;C.
 * 8&mu;L eluted DNA
 * 1&mu;L 10x Tango buffer
 * 1&mu;L XbaI

9. Purify the proper fragment in 3% agarose gel, elute DNA in 15&mu;L water using NucleoSpin Extract II DNA purification kit.

10. 5&mu;L small-scale ligation reaction Incubate 1 hr at room temperature. Include control ligation where DNA insert is replaced by water.
 * ~12ng digested plasmid backbone
 * ~5ng digested sRNA gene
 * 0.5&mu;L 10x T4 DNA Ligase Reaction Buffer
 * 0.5&mu;L T4 DNA ligase

11. Transform 2&mu;L of reaction into E. coli Top10F' cells. Expect 50-200 colonies.

Target-GFP Fusion Cloning

1. Inoculate single colony of E. coli Top10 cells containing pXG-10 plasmid into 4mL LB containing 20&mu;g/mL chloramphenicol, grow overnight at 37&deg;C with agitation.

2. Dilute culture in 400mL fresh LB medium, incubate overnight.

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 80&mu;L water.

4. 60&mu;L digestion reaction (pXG-10) Digest for 7 hr (or overnight) at 37&deg;C, then add 20&mu;L shrimp alkaline phosphatase (SAP) and incubate for 3 hr at 37&deg;C..
 * 4&mu;g pXG-10 plasmid
 * 40&mu;L NheI
 * 20&mu;L BfrBI
 * 1x Tango buffer

5. Load reaction on 1% agarose gel and excise the 4.1kbp band. Purify using NucleoSpin Extract II DNA purification kit and elute in 50&mu;L water.

6. 25&mu;L PCR reaction (sRNA target sequence) Run for 5 min @ 95&deg;C, then 30 cycles of the following: 45s @ 95&deg;C, 45s @ 58&deg;C, 45s @ 72&deg;C. Incubate for 5 min @ 72&deg;C, then analyze 5&mu;L of the reaction in 3% agarose gel.
 * 1&mu;L (10-50 ng) chromosomal E. coli K12 template DNA
 * 0.2&mu;L of each primer
 * 2.5&mu;L 10x Pfu buffer
 * 0.5&mu;L dNTP mix
 * 0.4&mu;L Pfu DNA polymerase
 * 20.2&mu;L water

7. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 15&mu;L water.

8. 15&mu;L digestion reaction (sRNA target sequence) Digest for 3 hr at 37&deg;C.
 * 12&mu;L eluted DNA
 * 7.5U NheI
 * 7.5U BfrBI
 * 1x Tango buffer

9. Purify the proper fragment in 3% agarose gel, elute DNA in 15&mu;L water using NucleoSpin Extract II DNA purification kit.

10. 5&mu;L small-scale ligation reaction Incubate 1 hr at room temperature. Include control ligation where DNA insert is replaced by water.
 * ~12ng digested pXG-10
 * ~5ng digested sRNA target sequence
 * 0.5&mu;L 10x T4 DNA Ligase Reaction Buffer
 * 0.5&mu;L T4 DNA ligase

11. Transform 2&mu;L of reaction into E. coli Top10 cells. Expect 50-200 colonies.