pNL4-3 vector (Cat. No.: V004299)

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Basic Information

Note: pNL4-3 (14,825 bp) is a full-length HIV-1 proviral DNA molecular clone widely used as a reference strain in AIDS research. It carries the entire HIV-1 NL4-3 genome, including all nine viral genes (gag, pol, env, tat, rev, nef, vif, vpr, vpu), flanked by 5‘ and 3’ cellular genomic sequences and flanking LTRs (Long Terminal Repeats) that control viral transcription and integration. The plasmid also encodes Protein Nef for studying viral pathogenesis. Upon transfection into suitable mammalian cells, pNL4-3 produces replication-competent HIV-1 virions, making it an essential tool for studying viral replication, drug resistance, and vaccine development. The vector backbone includes AmpR for bacterial selection and a pUC origin for high-copy replication in E. coli, typically grown at 30 °C in NEB Stable or Stbl3 cells to prevent LTR recombination

Name:: pNL4-3

Antibiotic Resistance:: Ampicillin

Length:: 14825 bp

Type:: HIV-1 vector

Replication origin:: ori

Source/Author:: Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA.

Copy Number:: High copy number

Growth Strain(s):: stbl3

Growth Temperature:: 30℃

$ 198.4

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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 sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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

1. Sharma PL, Crumpacker CS. Decreased processivity of human immunodeficiency virus type 1 reverse transcriptase (RT) containing didanosine-selected mutation Leu74Val: a comparative analysis of RT variants Leu74Val and lamivudine-selected Met184Val. J Virol. 1999 Oct;73(10):8448-56. doi: 10.1128/JVI.73.10.8448-8456.1999. PMID: 10482597; PMCID: PMC112864.

2. Kinomoto M, Yokoyama M, Sato H, Kojima A, Kurata T, Ikuta K, Sata T, Tokunaga K. Amino acid 36 in the human immunodeficiency virus type 1 gp41 ectodomain controls fusogenic activity: implications for the molecular mechanism of viral escape from a fusion inhibitor. J Virol. 2005 May;79(10):5996-6004. doi: 10.1128/JVI.79.10.5996-6004.2005. PMID: 15857986; PMCID: PMC1091722.