SRNA characterization procedure

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. 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.

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 proper 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.

Target-GFP Fusion Cloning

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.

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 80μL water.

4. 60μL digestion reaction (pXG-10)

• 4μg pXG-10 plasmid
• 40μL NheI
• 20μL BfrBI
• 1x Tango buffer

Digest for 7 hr (or overnight) at 37°C, then add 20μL shrimp alkaline phosphatase (SAP) and incubate for 3 hr at 37°C..

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.

6. 25μL PCR reaction (sRNA target sequence)

• 1μL (10-50 ng) chromosomal E. coli K12 template DNA
• 0.2μL of each primer
• 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 @ 58°C, 45s @ 72°C. Incubate for 5 min @ 72°C, then analyze 5μL of the reaction in 3% agarose gel.

7. Purify reaction with NucleoSpin Extract II DNA purification kit, elute DNA in 15μL water.

8. 15μL digestion reaction (sRNA target sequence)

• 12μL eluted DNA
• 7.5U NheI
• 7.5U BfrBI
• 1x Tango buffer

Digest for 3 hr at 37°C.

9. Purify the proper 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 pXG-10
• ~5ng digested sRNA target sequence
• 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 Top10 cells. Expect 50-200 colonies.