pHY300PLK vector (Cat. No.: V012462)

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Basic Information

Note: pHY300PLK functions as a Bacillus-Escherichia coli shuttle vector, mediating the cloning and expression of target genes to facilitate strain functional studies.

Name:: pHY300PLK

Antibiotic Resistance:: Ampicillin

Length:: 4872 bp

Type:: Cloning Vectors

Replication origin:: p15A ori

Source/Author:: TaKaRa

Copy Number:: Medium copy number

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

$ 199.4

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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.

References

Yao Z, Jeon HS, Yoo JY, Kang YJ, Kim MJ, Kim TJ, Kim JH. DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability. J Microbiol Biotechnol. 2022 Jun 28;32(6):800-807. doi: 10.4014/jmb.2202.02017. Epub 2022 Apr 25. PMID: 35484964; PMCID: PMC9628911.