pCDH-CMV-MCS-EF1-Puro vector (Cat. No.: V006738)

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Basic Information

Note: The pCDH-CMV-MCS-EF1-Puro is a lentiviral expression plasmid. It includes a CMV promoter for high-level expression of a gene cloned into its multiple cloning site (MCS) and an EF1 promoter for a puromycin resistance gene, enabling stable cell line generation

Name:: pCDH-CMV-MCS-EF1-Puro

Antibiotic Resistance:: Ampicillin

Length:: 7403 bp

Type:: Lentiviral vectors

Replication origin:: ori

Selection Marker:: Puromycin

Promoter:: RSV

Cloning Method:: Enzyme digestion and ligation

5' Primer:: CMV-F: CGC AAA TGG GCG GTA GGC GTG

$ 198.5

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

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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

Shuwen H, Yinhang W, Jing M, Gong C, Xiaohui H, Xi Y, Wei W. Open Reading Frame-3a gene of the 2019 novel coronavirus inhibits the occurrence and development of colorectal cancer. Discov Oncol. 2022 Mar 20;13(1):14. doi: 10.1007/s12672-022-00473-6. PMID: 35306605; PMCID: PMC8934246.

Fan T-J, Xie C, Li L, Jin X, Cui J, Wang J-H. HIV-1 Nef activates proviral DNA transcription by recruiting Src kinase to phosphorylate host protein Nef-associated factor 1 to compromise its viral restrictive function. J Virol. 2025 May 20;99(5):e0028025. doi: 10.1128/jvi.00280-25. Epub 2025 Apr 24. PMID: 40272155; PMCID: PMC12090801.