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Pegfp n1 plasmid

Manufactured by Takara Bio
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PEGFP-N1 plasmid is a commercially available vector designed for expression of proteins fused to the green fluorescent protein (GFP) in mammalian cells. The plasmid contains the GFP coding sequence under the control of a CMV promoter, allowing for robust expression of the GFP-fusion protein.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Nanodrop, by Thermo Fisher Scientific (4 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions) Endofree plasmid maxi kit, by Qiagen (3 mentions) Glutamine, by Thermo Fisher Scientific (3 mentions) No 1 coverglass, by MatTek (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Effectene reagent, by Qiagen (3 mentions) Pureyield plasmid midiprep kit, by Promega (2 mentions) Pshuttle cmv irfp plasmid, by Addgene (2 mentions) Rpmi 1640 medium, by Lonza (2 mentions) L glutamine, by Lonza (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Pcmv vsv g, by Addgene (1 mentions) Agarose gel, by Merck Group (1 mentions) Ligation kit, by Takara Bio (1 mentions) Hindiii, by Takara Bio (1 mentions) Pegfp n1, by Merck Group (1 mentions) Te 2000s fluorescence microscope, by Nikon (1 mentions)

Close spelling variants of this product

PEGFP-N1 plasmid vector (2 citations) PEGFP-N1 (560 citations) PEGFP plasmid (8 citations)

66 protocols using pegfp n1 plasmid

1

Maintenance and Manipulation of Chicken DT40 and Jurkat Cells

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Culture maintenance, plasmid transfection and BCR stimulation of chicken DT40 B cell lines were carried out as previously described46 (link). Jurkat cell culture and transfection techniques were also performed as described8 (link). The SOS1−2−deficient DT40 B cells were generated in Dr. Tomohiro Kurosaki’s laboratory (RIKEN). Both wildtype and SOS1−2− deficient DT40 B cells were gifts from Dr. Kurosaki. Obtained cell lines were confirmed to be free of mycoplasma contamination. For routine cell functional authentication, surface expression of B cell receptor (BCR) was confirmed by flow cytometry and by BCR-induced pERK2 measurement similar to the experiment shown in Supplementary Figure 4. Jurkat T cells were obtained from ACCC and were maintained according to the provided guideline.
To generate EGFP-tagged hSOS1 variants, EGFP coding sequence (CDS) was PCR-amplified with Xba I- and Not I-flanked primers from pEGFP-N1 plasmid (Clonetech). Resulting SOS1-EGFP construct bears a 5 amino acid linker (SRGGR) between SOS1 and EGFP CDS. Expression was confirmed by Western blotting with anti-GFP antibody (Supplementary Fig. 4a).
Christensen S.M., Tu H.L., Jun J.E., Alvarez S., Triplet M.G., Iwig J.S., Yadav K.K., Bar-Sagi D., Roose J.P, & Groves J.T. (2016). One-way membrane trafficking of SOS in receptor-triggered Ras activation. Nature structural & molecular biology, 23(9), 838-846.
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Maintenance and Manipulation of Chicken DT40 and Jurkat Cells

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Culture maintenance, plasmid transfection and BCR stimulation of chicken DT40 B cell lines were carried out as previously described46 (link). Jurkat cell culture and transfection techniques were also performed as described8 (link). The SOS1−2−deficient DT40 B cells were generated in Dr. Tomohiro Kurosaki’s laboratory (RIKEN). Both wildtype and SOS1−2− deficient DT40 B cells were gifts from Dr. Kurosaki. Obtained cell lines were confirmed to be free of mycoplasma contamination. For routine cell functional authentication, surface expression of B cell receptor (BCR) was confirmed by flow cytometry and by BCR-induced pERK2 measurement similar to the experiment shown in Supplementary Figure 4. Jurkat T cells were obtained from ACCC and were maintained according to the provided guideline.
To generate EGFP-tagged hSOS1 variants, EGFP coding sequence (CDS) was PCR-amplified with Xba I- and Not I-flanked primers from pEGFP-N1 plasmid (Clonetech). Resulting SOS1-EGFP construct bears a 5 amino acid linker (SRGGR) between SOS1 and EGFP CDS. Expression was confirmed by Western blotting with anti-GFP antibody (Supplementary Fig. 4a).
Christensen S.M., Tu H.L., Jun J.E., Alvarez S., Triplet M.G., Iwig J.S., Yadav K.K., Bar-Sagi D., Roose J.P, & Groves J.T. (2016). One-way membrane trafficking of SOS in receptor-triggered Ras activation. Nature structural & molecular biology, 23(9), 838-846.
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3

Cellular Expression of HIV-1 Nef Protein

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For cellular expression of Nef, h-astrocytes were transfected with pEGFP-N1 plasmid (Takara Bio USA) coding for green fluorescent protein (GFP; pGFP) or HIV-1 SF2 Nef C-terminally tagged with GFP (pNef.GFP; NIH AIDS Reagent Program). For production of pseudotyped HIV-1 viral stocks, HEK293T were transfected with plasmids coding for HIV-1 isolates NL4-3, NL4-3 Δnef, or YU-2 (obtained through NIH AIDS Reagent Program), and a plasmid coding for vesicular stomatitis virus envelope protein (VSV-G; pCMV-VSV-G; Addgene).
Dominkuš P.P., Ferdin J., Plemenitaš A., Peterlin B.M, & Lenassi M. (2017). Nef is secreted in exosomes from Nef.GFP-expressing and HIV-1-infected human astrocytes. Journal of neurovirology, 23(5), 713-724.
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4

Cloning 6xMycTag-SV40polyA and EGFP

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6xMycTag-SV40polyA fragment was amplified from pCS2+nlsMT plasmid (a gift from R. Rupp, Adolf-Buteland Institute, Munich, Germany) and cloned in the XbaI site of the pattB vector (Bischof et al., 2013 (link)). EGFP sequence was amplified from pEGFP-N1 plasmid (Takara Bio Inc.) and cloned in the KpnI site of the previous construct, in frame with the 6xMycTag sequence.
Vieillard J., Paschaki M., Duteyrat J.L., Augière C., Cortier E., Lapart J.A., Thomas J, & Durand B. (2016). Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells. The Journal of Cell Biology, 214(7), 875-889.
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5

Cloning Tomato Sequence with SV40polyA

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The Tomato sequence was obtained by PCR on the Zeo-TomatoNT-2 (a gift from R. Basto, Institut Curie, Paris, France) and further cloned in the KpnI site of the pattB plasmid. SV40polyA was amplified from pEGFP-N1 plasmid (Takara Bio Inc.) and cloned in the XbaI site of the previous plasmid.
Vieillard J., Paschaki M., Duteyrat J.L., Augière C., Cortier E., Lapart J.A., Thomas J, & Durand B. (2016). Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells. The Journal of Cell Biology, 214(7), 875-889.
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6

Cloning of PHB1 into pEGFP-N1 Plasmid

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The coding region of PHB1 was amplified by PCR, digested with Hind III and Kpn I (TAKARA, Japan), and directly cloned and inserted into the pEGFP-N1 plasmid (TAKARA, Japan) to generate the pEGFP-PHB1 plasmid. Briefly, the coding region of PHB1 (819 bp) was amplified using high-fidelity PCR (Thermo Fisher Scientific, United States). The coding regions of PHB1 and pEGFP-N1 were digested with the restriction enzymes Hind III and Kpn I. The digestion products were examined by electrophoresis on a 1% (w/v) agarose gel (Sigma-Aldrich, United States). Ligation reactions (Ligation Kit, TAKARA, Japan) were performed to join the DNA fragments, which were subsequently transformed into competent Escherichia coli cells (E. coli Top 10, TAKARA, Japan) at 37 °C overnight (12-16 h). Colony selection was performed by PCR, and the amplicons were examined by electrophoresis using a 1% (w/v) agarose gel. Plasmid extraction from positive colonies was performed with an E.Z.N.A.® Endo-Free Plasmid Midi Kit (Omega, GA, United States), and the sequences were verified by sequencing. The primer pair that was used to amplify PHB1 was 5’-TGGGAGGTCTATATAAGCAGAG-3’ and 5’-CGTCGCCGTCCAGCTCG ACCAG-3’.
Shi J.J., Wang Y.K., Wang M.Q., Deng J., Gao N., Li M., Li Y.P., Zhang X., Jia X.L., Liu X.T., Dang S.S, & Wang W.J. (2024). Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro. World Journal of Gastrointestinal Oncology, 16(2), 398-413.
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7

Transient GFP Expression in EK Cells

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The pEGFP-N1 plasmid (Takara) expressing a green fluorescent protein (GFP) was used for cell transfection. The EK cells at passage 60 were inoculated at a density of 1×105 cells well−1 in a 6-well plate. After 48 h, the cells were transfected with 5 μg pEGFP-N1 plasmid in 12 μl lipofectamine™ 2000 reagent (Takara) and incubated at 28°C for 8 h. The cell culture was then transferred to L-15 supplemented with 10% FBS. After 18 h, the fluorescence signals from the cells were observed by a Nikon TE2000S fluorescence microscope, the transfection efficiency was calculated by counting the ratio of GFP-positive cells to all cells in 10 different optical fields.
Chen B., Zheng Z., Yang J., Chi H., Huang H, & Gong H. (2018). Development and characterization of a new cell line derived from European eel Anguilla anguilla kidney. Biology Open, 8(1), bio037507.
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8

Construction of pdAb3FLAG Expression Vector

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The construction of the IRES-containing αCD3 × αCEA diabody expression vector pdAb3 has been previously described.11 (link) To construct the pdAb3FLAG expression vector the pdAb3 plasmid was digested with NheI/BlpI to remove the cytomegalovirus enhancer/promoter, the heterologous oncostation M signal peptide and the partial VH MFE23 domain. A synthetic gene encoding the cytomegalovirus enhancer/promoter, the oncostation M signal peptide, a N-terminal FLAG tag (DYKDDDDK) and the partial VH domain of MFE23 was synthesized by Geneart AG (Life Technologies) and cloned using NheI/BlpI restriction sites. The IRES sequence was removed using NotI/XhoI restriction sites and a synthetic gene encoding the 2A sequence derived from FMDV and the diabody chain 2 (VH-OKT3 and VLMFE23) along with the C-terminal myc-tag and 6xHis-tag was synthesized by Geneart AG and cloned to construct the pdAb3FLAG-F2A expression vector. The pEGFP-N1 plasmid (Takara Bio Inc., Shiga, Japan) was used for evaluating transfection efficiency.
Mølgaard K., Compte M., Nuñez-Prado N., Harwood S.L., Sanz L, & Alvarez-Vallina L. (2017). Balanced secretion of anti-CEA × anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity. Gene Therapy, 24(4), 208-214.
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9

Transfection and Subcellular Localization

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Twenty-four hours prior to transfection, the cells were seeded onto 12-well plates. For transfections, 450 ng of the respective AGA expression plasmid and 50 ng of pRL-TK encoding renilla luciferase for normalization (Promega, Mannheim, Germany) were transfected using MACSfectin™ (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. After 24 h, the cells were transferred into 6-well plates and harvested 48 h post-transfection using 250 µL passive lysis buffer (Promega). Fibroblasts were electroporated using the Neon™ Transfection System (Invitrogen) according to the manufacturer’s instruction. 1 × 106 cells in a total volume of 100 µL were transfected with 1 µg plasmid (1200 V, 40 ms, 1 pulse). Cells that were used for lysotracker stainings were cotransfected with 250 ng pEGFP-N1 plasmid (Clontech, Takara, Saint-Germain-en-Laye, France) for the detection of transfected cells. Transfected cells were either seeded into 6 well plates and used for enzyme activity measurements or onto coverslips and used for staining with Lysotracker as described [20 (link)].
Banning A., König J.F., Gray S.J, & Tikkanen R. (2017). Functional Analysis of the Ser149/Thr149 Variants of Human Aspartylglucosaminidase and Optimization of the Coding Sequence for Protein Production. International Journal of Molecular Sciences, 18(4), 706.
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10

Plasmid Transformation and Purification

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pEGFP-N1 plasmid (Clontech, Mountain View, California, US) was transformed into XL1-Blue [dut+, ung+] (Stratagene, San Diego, California (CA), US) or CJ236 [dut-, ung-] (NEB) E. coli competent cells. Cell cultures were grown for 16 hr in Luria broth (LB) media supplemented with 50 µg/ml kanamycin at 37°C. Plasmids used in this study were purified using PureYield Plasmid Midiprep Kit (Promega, Madison, Wisconsin, US) according to the instructions of the manufacturer. XL1-Blue and CJ236 E. coli strains were propagated in LB media at 37°C and were harvested at log phase. Genomic DNA of bacterial samples as well as eukaryote cells was purified using the Quick-DNA Miniprep Plus Kit (Zymo Research, Irvine, California, US) using the recommendations of the manufacturer.
Pálinkás H.L., Békési A., Róna G., Pongor L., Papp G., Tihanyi G., Holub E., Póti Á., Gemma C., Ali S., Morten M.J., Rothenberg E., Pagano M., Szűts D., Győrffy B, & Vértessy B.G. (2020). Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments. eLife, 9, e60498.
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