pENTR4 vector (Cat. No.: V011809)

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Basic Information

Note: The pENTR4 is a 3.7 kb Gateway entry vector for cloning genes flanked by attL1 and attL2 sites. It is a high-copy plasmid with a kanamycin resistance gene and a pUC origin. A key feature is the ccdB lethal gene between the attL sites, requiring the use of ccdB-resistant strains like DB3.1 for propagation. Cloned inserts can be transferred via LR recombination to destination vectors for expression studies.

Name:: pENTR4

Antibiotic Resistance:: Kanamycin

Length:: 3757 bp

Type:: Gateway Cloning Vectors

Replication origin:: ori

Source/Author:: Invitrogen (Life Technologies)

Copy Number:: High copy number

Promoter:: lac UV5

Growth Strain(s):: DB3.1

Growth Temperature:: 37℃

$ 192.8

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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.

References

Chen C, Masi R, Lintermann R, Wirthmueller L. Nuclear Import of Arabidopsis Poly(ADP-Ribose) Polymerase 2 Is Mediated by Importin-α and a Nuclear Localization Sequence Located Between the Predicted SAP Domains. Front Plant Sci. 2018 Nov 1;9:1581. doi: 10.3389/fpls.2018.01581. PMID: 30455710; PMCID: PMC6230994.