pNZ8148 vector (Cat. No.: V012804)

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Basic Information

Note: pNZ8148 is a chloramphenicol-resistant plasmid. It includes 2 genes for the replication proteins repA and repC, and the nisin-inducible promoter (P nisA). The replicons of the vectors pNZ8008, pNZ8148, pNZ8149 and pNZ8150 are identical and came originally from the Lactococcus lactis plasmid pSH71. However, this replicon has a broad host range. Plasmids with this replicon can replicate in many Gram-positive bacteria, such as Lactobacillus plantarum and Streptococcus thermophilus. For propagation of pNZ8148, we recommend E. coli strain MC1061 (10 µg/ml chloramphenicol; growth of colonies may take 2 days). pUC57-pNZ8148 is recommended as a replacement, which can be grown in conventional clonal strains and is easier to use.

Name:: pNZ8148

Antibiotic Resistance:: Chloramphenicol

Length:: 3167 bp

Type:: expression plasmid of Lactobacilli

Promoter:: PnisA

Cloning Method:: Restriction Enzyme

Growth Strain(s):: MC1061

Growth Temperature:: 30℃

$ 197.8

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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 20 μl of sterile water to dissolve the DNA.

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.

Note: Use MC1061 competent cells; 10 μg/ml chloramphenicol as antibiotics; Grow the colonies at 30°C and it may take 2 days.

References

Kazemi-Roudsari, Mahsa et al. “Design of an oral vaccine using Lactococcus lactis against brucellosis: an in vitro and in vivo study.” AMB Express vol. 14,1 2. 3 Jan. 2024, doi:10.1186/s13568-023-01638-4

Fu, Rui-Yan et al. Wei sheng wu xue bao = Acta microbiologica Sinica vol. 46,3 (2006): 379-84.