pKD3 vector (Cat. No.: V012131)

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Basic Information

Note: The pKD3 plasmid is used for gene knockout via Red/ET recombination in E. coli. It contains an FRT-flanked cassette for marker removal.

Name:: pKD3

Antibiotic Resistance:: Ampicillin

Length:: 2805 bp

Type:: Knockout Vectors

Replication origin:: R6K γ ori

Promoter:: R6K

Cloning Method:: Enzyme digestion and ligation

5' Primer:: R6K-3

3' Primer:: rrnB-T1-term-F

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

$ 199.6

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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

Zou Z, Zeng F, Xu W, Wang C, Ke Z, Wang QJ, Deng F. PKD2 and PKD3 promote prostate cancer cell invasion by modulating NF-κB- and HDAC1-mediated expression and activation of uPA. J Cell Sci. 2012 Oct 15;125(Pt 20):4800-11. doi: 10.1242/jcs.106542. Epub 2012 Jul 13. PMID: 22797919; PMCID: PMC4074284.