pCold TF vector (Cat. No.: V008303)

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Basic Information

Note: pCold TF is a cold shock protein expression vector. It utilizes the cspA promoter for induction at low temperatures and fuses the target protein to a Trigger Factor (TF) chaperone to enhance solubility and correct folding in E. coli. It includes tags for purification and protease sites for tag removal.This vector inserts a 5' non-coding region (5'UTR), TEE (Translation Enhancement Element) sequence, His-Tag sequence, TF sequence, and Multiple Cloning Site (MCS) downstream of the cspA promoter. A lac operator was also inserted downstream of the cspA promoter to tightly regulate protein expression. And cut-off sites for HRV 3C Protease, Thrombin and Factor Xa are inserted between the TF sequence and MCS, which are used to eliminate tags of fusion proteins. pCold TF DNA applies the promoter of E. coli, and like the other pCold DNAs, most of E. coli can be used as an expression host. Using pCold TF DNA, soluble expression of difficult-to-express genes can be achieved through the soluble tagging function of TF and the molecular chaperone effect.

Name:: pCold TF

Antibiotic Resistance:: Ampicillin

Length:: 5769 bp

Type:: Expression vector

Replication origin:: ori

Source/Author:: Shodai T, Takakura H.

Growth Temperature:: 37℃

$ 199.3

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Sui B, Wang X, Zhao T, Zhen J, Ren H, Liu W, Zhang X, Zhang C. Design, Screening, and Characterization of Engineered Phage Endolysins with Extracellular Antibacterial Activity against Gram-Negative Bacteria. Appl Environ Microbiol. 2023 Jul 26;89(7):e0058123. doi:10.1128/aem.00581-23. Epub 2023 Jun 20. PMID:37338346; PMCID: PMC10370328.