Structured Review

Eurofins plasmid dna
Plasma membrane targeting of SSTR3 mutants. (A) The indicated SSTR3 constructs, all containing C-terminal EGFP, were expressed in <t>IMCD3</t> cells and analyzed by immunofluorescence with antibodies against EGFP (green), ARL13B (red) and γ-tubulin (γTub, magenta). Cells were also stained with DAPI <t>(DNA).</t> All six constructs clearly reach the plasma membrane. (A, B) Indicated constructs were analyzed as in (A). IC3-mut1+CT-D335-428 mutant, lacking all but the first 10 residues in SSTR3-CT, consistently accumulates intracellularly and fails to reach plasma and ciliary membranes (left panels). In contrast, CT-mut1+IC3-D5, despite its intracellular retention frequency being much higher than normal (middle panels and Fig 7K ), can still reach ciliary and/or plasma membrane in about 60% of transfected cells (right panels). Scale bars, 5 μm.
Plasmid Dna, supplied by Eurofins, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "HTR6 and SSTR3 ciliary targeting relies on both IC3 loops and C-terminal tails"

Article Title: HTR6 and SSTR3 ciliary targeting relies on both IC3 loops and C-terminal tails

Journal: Life Science Alliance

doi: 10.26508/lsa.202000746

Plasma membrane targeting of SSTR3 mutants. (A) The indicated SSTR3 constructs, all containing C-terminal EGFP, were expressed in IMCD3 cells and analyzed by immunofluorescence with antibodies against EGFP (green), ARL13B (red) and γ-tubulin (γTub, magenta). Cells were also stained with DAPI (DNA). All six constructs clearly reach the plasma membrane. (A, B) Indicated constructs were analyzed as in (A). IC3-mut1+CT-D335-428 mutant, lacking all but the first 10 residues in SSTR3-CT, consistently accumulates intracellularly and fails to reach plasma and ciliary membranes (left panels). In contrast, CT-mut1+IC3-D5, despite its intracellular retention frequency being much higher than normal (middle panels and Fig 7K ), can still reach ciliary and/or plasma membrane in about 60% of transfected cells (right panels). Scale bars, 5 μm.
Figure Legend Snippet: Plasma membrane targeting of SSTR3 mutants. (A) The indicated SSTR3 constructs, all containing C-terminal EGFP, were expressed in IMCD3 cells and analyzed by immunofluorescence with antibodies against EGFP (green), ARL13B (red) and γ-tubulin (γTub, magenta). Cells were also stained with DAPI (DNA). All six constructs clearly reach the plasma membrane. (A, B) Indicated constructs were analyzed as in (A). IC3-mut1+CT-D335-428 mutant, lacking all but the first 10 residues in SSTR3-CT, consistently accumulates intracellularly and fails to reach plasma and ciliary membranes (left panels). In contrast, CT-mut1+IC3-D5, despite its intracellular retention frequency being much higher than normal (middle panels and Fig 7K ), can still reach ciliary and/or plasma membrane in about 60% of transfected cells (right panels). Scale bars, 5 μm.

Techniques Used: Construct, Immunofluorescence, Staining, Mutagenesis, Transfection

CDK5 phosphorylation of Ser-352 in HTR6-CT does not affect HTR6 ciliary targeting. (A) IMCD3 cells expressing C-terminally EGFP-tagged Chimera J with the S352A or S352D mutations were analyzed by immunofluorescence with antibodies against EGFP (green), ARL13B (red) and γ-tubulin (γTub, magenta). DNA was stained with DAPI. Arrows indicate cilia. Scale bar, 5 μm. Serine-352 is the mouse HTR6 equivalent of human HTR6 Serine-350, shown to be a target of CDK5 phosphorylation ( 17 ). S352A and S352D are non-phosphorylatable and phosphomimetic S352 mutants, respectively. (A, B) Quantification of ciliary localization from (A). Data are mean ± SEM of n = 3 independent experiments per construct, in each of which at least 50 transfected-cell cilia were counted for each G protein-coupled receptor. No significant differences were found by one-way ANOVA.
Figure Legend Snippet: CDK5 phosphorylation of Ser-352 in HTR6-CT does not affect HTR6 ciliary targeting. (A) IMCD3 cells expressing C-terminally EGFP-tagged Chimera J with the S352A or S352D mutations were analyzed by immunofluorescence with antibodies against EGFP (green), ARL13B (red) and γ-tubulin (γTub, magenta). DNA was stained with DAPI. Arrows indicate cilia. Scale bar, 5 μm. Serine-352 is the mouse HTR6 equivalent of human HTR6 Serine-350, shown to be a target of CDK5 phosphorylation ( 17 ). S352A and S352D are non-phosphorylatable and phosphomimetic S352 mutants, respectively. (A, B) Quantification of ciliary localization from (A). Data are mean ± SEM of n = 3 independent experiments per construct, in each of which at least 50 transfected-cell cilia were counted for each G protein-coupled receptor. No significant differences were found by one-way ANOVA.

Techniques Used: Expressing, Immunofluorescence, Staining, Construct, Transfection

Plasma membrane targeting of HTR6 mutants. The indicated HTR6 constructs, all containing C-terminal EGFP, were expressed in IMCD3 cells, which were analyzed by immunofluorescence with antibodies against EGFP (green), acetylated tubulin (AcTub, left) or ARL13B (right) (red) and gamma-tubulin (γTub, magenta). Cells were also stained with DAPI (DNA). All HTR6 constructs clearly label the plasma membrane. Scale bar, 5 μm.
Figure Legend Snippet: Plasma membrane targeting of HTR6 mutants. The indicated HTR6 constructs, all containing C-terminal EGFP, were expressed in IMCD3 cells, which were analyzed by immunofluorescence with antibodies against EGFP (green), acetylated tubulin (AcTub, left) or ARL13B (right) (red) and gamma-tubulin (γTub, magenta). Cells were also stained with DAPI (DNA). All HTR6 constructs clearly label the plasma membrane. Scale bar, 5 μm.

Techniques Used: Construct, Immunofluorescence, Staining

HTR6 ciliary targeting requires the ciliary trafficking adapter TULP3. (A) HTR6-IMCD3 cells were transiently transfected with siRNAs targeting mouse TULP3 (siTULP3 #1 or siTULP3 #2) or firefly luciferase (siLUC) as negative control, serum-starved to promote ciliogenesis and immunostained with anti-ARL13B (green) and anti-HTR6 (red) antibodies. DNA was stained with Hoechst (blue). Scale bar, 5 μm. (A, B) HTR6 ciliary intensity was quantified from (A). Data are mean ± SEM of n = 23,32,29 cells for siLUC, siTULP3 #1, and siTULP3 #2, respectively. (C) Mouse Tulp3 mRNA levels were analyzed by qRT-PCR and expressed relative to Gapdh mRNA. Data are mean ± SEM of n = 3 independent experiments. Significance in (B, C) is shown as P
Figure Legend Snippet: HTR6 ciliary targeting requires the ciliary trafficking adapter TULP3. (A) HTR6-IMCD3 cells were transiently transfected with siRNAs targeting mouse TULP3 (siTULP3 #1 or siTULP3 #2) or firefly luciferase (siLUC) as negative control, serum-starved to promote ciliogenesis and immunostained with anti-ARL13B (green) and anti-HTR6 (red) antibodies. DNA was stained with Hoechst (blue). Scale bar, 5 μm. (A, B) HTR6 ciliary intensity was quantified from (A). Data are mean ± SEM of n = 23,32,29 cells for siLUC, siTULP3 #1, and siTULP3 #2, respectively. (C) Mouse Tulp3 mRNA levels were analyzed by qRT-PCR and expressed relative to Gapdh mRNA. Data are mean ± SEM of n = 3 independent experiments. Significance in (B, C) is shown as P

Techniques Used: Transfection, Luciferase, Negative Control, Staining, Quantitative RT-PCR

HTR6 labels cilia not only more frequently but also more intensely than HTR7. (A) IMCD3 cells expressing HTR6-EGFP or HTR7-EGFP were analyzed by immunofluorescence with antibodies against EGFP (green) and ARL13B (red). DNA was stained with DAPI (blue). Scale bar, 10 μm. (A, B) Quantitation of ciliary intensity of G protein-coupled receptor-positive cilia from (A). Cilia showing no detectable G protein-coupled receptor staining were not included in the analysis (as shown in Figs 2 and 3 , HTR7 only labels about 20% of cilia, whereas HTR6 labels virtually 100%). Data are mean ± SEM of n = 42 (HTR6) and n = 31 (HTR7) cilia. Significance is shown as P
Figure Legend Snippet: HTR6 labels cilia not only more frequently but also more intensely than HTR7. (A) IMCD3 cells expressing HTR6-EGFP or HTR7-EGFP were analyzed by immunofluorescence with antibodies against EGFP (green) and ARL13B (red). DNA was stained with DAPI (blue). Scale bar, 10 μm. (A, B) Quantitation of ciliary intensity of G protein-coupled receptor-positive cilia from (A). Cilia showing no detectable G protein-coupled receptor staining were not included in the analysis (as shown in Figs 2 and 3 , HTR7 only labels about 20% of cilia, whereas HTR6 labels virtually 100%). Data are mean ± SEM of n = 42 (HTR6) and n = 31 (HTR7) cilia. Significance is shown as P

Techniques Used: Expressing, Immunofluorescence, Staining, Quantitation Assay

Membrane association is needed for HTR6-CT to function as a ciliary targeting sequence. (A) IMCD3 cells expressing C-terminally EGFP-tagged HTR6-CT were analyzed by immunofluorescence with antibodies against EGFP (green), acetylated tubulin (AcTub, red) and γ-tubulin (γTub, magenta). DNA was stained with DAPI. Arrows indicate cilia. Scale bar, 5 μm. (A, B) Quantification of ciliary localization from (A). Data are mean ± SEM of n = 5 and n = 3 independent experiments for CD8α(1-206)-CT(HTR6)-EYFP and CT(HTR6)-EGFP, respectively. In each experiment, at least 50 transfected-cell cilia were counted for each construct. Significance in unpaired two-tailed t test is shown as P
Figure Legend Snippet: Membrane association is needed for HTR6-CT to function as a ciliary targeting sequence. (A) IMCD3 cells expressing C-terminally EGFP-tagged HTR6-CT were analyzed by immunofluorescence with antibodies against EGFP (green), acetylated tubulin (AcTub, red) and γ-tubulin (γTub, magenta). DNA was stained with DAPI. Arrows indicate cilia. Scale bar, 5 μm. (A, B) Quantification of ciliary localization from (A). Data are mean ± SEM of n = 5 and n = 3 independent experiments for CD8α(1-206)-CT(HTR6)-EYFP and CT(HTR6)-EGFP, respectively. In each experiment, at least 50 transfected-cell cilia were counted for each construct. Significance in unpaired two-tailed t test is shown as P

Techniques Used: Sequencing, Expressing, Immunofluorescence, Staining, Transfection, Construct, Two Tailed Test

Related Articles

Plasmid Preparation:

Article Title: Regulatory polymorphisms in the bovine Ankyrin 1 gene promoter are associated with tenderness and intramuscular fat content
Article Snippet: The quality and quantity of the purified PCR product was assessed on a 1.2% agarose gel and on a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc. MA, USA). .. Randomly selected 12 plasmid DNAs (200 ng) were sequenced in both reverse and forward directions to confirm constructs by Eurofins, MWG-Biotech (Edelsberg, Germany). ..

Article Title: Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)
Article Snippet: Klenow fragment, and cloned into SmaI site of pCMV and pCMVFlag vectors. .. These plasmid DNAs, verified by DNA sequencing (Eurofins), were transiently transfected into HEK293 cells by electroporation (X-Cell, Bio-Rad) as described previously [ ]. .. Human Fpn1 siRNA was purchased from Sigma-Aldrich ( GAUGGAACUUGGUAUCCAU [dT][dT], and AUGGAUACCAAGUUCCAUC[dT][dT]) and 5ul of 20uM solution was transfected into Caco-2 cells (grown in 2ml media/35 mm plate) with 200ul of Opti-MEM containing 6ul of Lipofectamine RNAiMax (Invitrogen) for 18hr, followed by treatment with 250 and 500uM FAC for additional 8hr in the presence of siRNA prior to harvest.

Article Title: Residues of acidic chitinase cause chitinolytic activity degrading chitosan in porcine pepsin preparations
Article Snippet: Each amplified DNA was then digested with EcoRI and XhoI and subcloned into the pEZZ18 expression vector. .. The entire nucleotide sequence of the resulting plasmid DNAs (pEZZ18/PA-CatD, pEZZ18/PA-CatDΔ21 or PA-CatDΔ46) was confirmed by sequencing (Eurofins Genomics, Tokyo, Japan). .. The recombinant PA-Chia, PA-CatD, PA-CatDΔ21 and PA-CatDΔ46 (Supplementary Fig. ) were prepared as described previously , .

Article Title: The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80*, and FAM155A* constructs (figs. S3 to S5) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genomics). .. For expression in X. laevis oocytes, plasmid DNAs were linearized with Xba I restriction enzyme, from which capped RNAs were synthesized using the T7 mMessage mMachine Kit (Ambion).

Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation
Article Snippet: All chemicals used for ORi preparation were purchased from Fluka (Deisenhofen, Germany), except for HEPES, penicillin, and streptomycin (Sigma-Aldrich). .. Plasmid DNAs encoding the human CHRNA9 and CHRNA10 as well as the 43 kDa receptor-associated protein of the synapse ( RAPSN ) were obtained from Eurofins Genomics (Ebersberg, Germany) and capped cRNA was synthesized as described before ( ). .. Human CHRNA7 encoding cRNA was kindly provided by G. Schmalzing (Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany) and synthesized as described before ( ). cRNA was dissolved in nuclease-free water and injected into oocytes in a volume of 50.6 nl using a microinjector (Nanoject, Drummond Scientific, Broomall, PA, USA).

Article Title: The sodium leak channel complex is modulated by voltage and extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80* and FAM155A* constructs ( and ) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genonimcs). .. For expression in Xenopus laevis oocytes, plasmid DNAs were linearized with XbaI restriction enzyme, from which capped RNAs were synthesised using the T7 mMessage mMachine Kit (Ambion).

Article Title: Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA
Article Snippet: After heating to 95 °C (to switch from the RT reaction to the PCR, following the manufacturer’s instructions), the reverse primer was added (5′-GTCATTCTCCTAAGAAGC-3′). .. Cloning of the PCR products was performed with the CloneJET PCR Cloning Kit (Thermo Fisher Scientific), and plasmid DNAs were sequenced using a third primer (5′-CGATGAGTTTTCGGTATTATC-3′) by Eurofins Genomics. ..

Construct:

Article Title: Regulatory polymorphisms in the bovine Ankyrin 1 gene promoter are associated with tenderness and intramuscular fat content
Article Snippet: The quality and quantity of the purified PCR product was assessed on a 1.2% agarose gel and on a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc. MA, USA). .. Randomly selected 12 plasmid DNAs (200 ng) were sequenced in both reverse and forward directions to confirm constructs by Eurofins, MWG-Biotech (Edelsberg, Germany). ..

DNA Sequencing:

Article Title: Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)
Article Snippet: Klenow fragment, and cloned into SmaI site of pCMV and pCMVFlag vectors. .. These plasmid DNAs, verified by DNA sequencing (Eurofins), were transiently transfected into HEK293 cells by electroporation (X-Cell, Bio-Rad) as described previously [ ]. .. Human Fpn1 siRNA was purchased from Sigma-Aldrich ( GAUGGAACUUGGUAUCCAU [dT][dT], and AUGGAUACCAAGUUCCAUC[dT][dT]) and 5ul of 20uM solution was transfected into Caco-2 cells (grown in 2ml media/35 mm plate) with 200ul of Opti-MEM containing 6ul of Lipofectamine RNAiMax (Invitrogen) for 18hr, followed by treatment with 250 and 500uM FAC for additional 8hr in the presence of siRNA prior to harvest.

Article Title: The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80*, and FAM155A* constructs (figs. S3 to S5) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genomics). .. For expression in X. laevis oocytes, plasmid DNAs were linearized with Xba I restriction enzyme, from which capped RNAs were synthesized using the T7 mMessage mMachine Kit (Ambion).

Article Title: The sodium leak channel complex is modulated by voltage and extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80* and FAM155A* constructs ( and ) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genonimcs). .. For expression in Xenopus laevis oocytes, plasmid DNAs were linearized with XbaI restriction enzyme, from which capped RNAs were synthesised using the T7 mMessage mMachine Kit (Ambion).

Transfection:

Article Title: Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)
Article Snippet: Klenow fragment, and cloned into SmaI site of pCMV and pCMVFlag vectors. .. These plasmid DNAs, verified by DNA sequencing (Eurofins), were transiently transfected into HEK293 cells by electroporation (X-Cell, Bio-Rad) as described previously [ ]. .. Human Fpn1 siRNA was purchased from Sigma-Aldrich ( GAUGGAACUUGGUAUCCAU [dT][dT], and AUGGAUACCAAGUUCCAUC[dT][dT]) and 5ul of 20uM solution was transfected into Caco-2 cells (grown in 2ml media/35 mm plate) with 200ul of Opti-MEM containing 6ul of Lipofectamine RNAiMax (Invitrogen) for 18hr, followed by treatment with 250 and 500uM FAC for additional 8hr in the presence of siRNA prior to harvest.

Electroporation:

Article Title: Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin)
Article Snippet: Klenow fragment, and cloned into SmaI site of pCMV and pCMVFlag vectors. .. These plasmid DNAs, verified by DNA sequencing (Eurofins), were transiently transfected into HEK293 cells by electroporation (X-Cell, Bio-Rad) as described previously [ ]. .. Human Fpn1 siRNA was purchased from Sigma-Aldrich ( GAUGGAACUUGGUAUCCAU [dT][dT], and AUGGAUACCAAGUUCCAUC[dT][dT]) and 5ul of 20uM solution was transfected into Caco-2 cells (grown in 2ml media/35 mm plate) with 200ul of Opti-MEM containing 6ul of Lipofectamine RNAiMax (Invitrogen) for 18hr, followed by treatment with 250 and 500uM FAC for additional 8hr in the presence of siRNA prior to harvest.

Sequencing:

Article Title: Residues of acidic chitinase cause chitinolytic activity degrading chitosan in porcine pepsin preparations
Article Snippet: Each amplified DNA was then digested with EcoRI and XhoI and subcloned into the pEZZ18 expression vector. .. The entire nucleotide sequence of the resulting plasmid DNAs (pEZZ18/PA-CatD, pEZZ18/PA-CatDΔ21 or PA-CatDΔ46) was confirmed by sequencing (Eurofins Genomics, Tokyo, Japan). .. The recombinant PA-Chia, PA-CatD, PA-CatDΔ21 and PA-CatDΔ46 (Supplementary Fig. ) were prepared as described previously , .

Purification:

Article Title: The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80*, and FAM155A* constructs (figs. S3 to S5) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genomics). .. For expression in X. laevis oocytes, plasmid DNAs were linearized with Xba I restriction enzyme, from which capped RNAs were synthesized using the T7 mMessage mMachine Kit (Ambion).

Article Title: The sodium leak channel complex is modulated by voltage and extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80* and FAM155A* constructs ( and ) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genonimcs). .. For expression in Xenopus laevis oocytes, plasmid DNAs were linearized with XbaI restriction enzyme, from which capped RNAs were synthesised using the T7 mMessage mMachine Kit (Ambion).

Transformation Assay:

Article Title: The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80*, and FAM155A* constructs (figs. S3 to S5) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genomics). .. For expression in X. laevis oocytes, plasmid DNAs were linearized with Xba I restriction enzyme, from which capped RNAs were synthesized using the T7 mMessage mMachine Kit (Ambion).

Article Title: The sodium leak channel complex is modulated by voltage and extracellular calcium
Article Snippet: Truncated NALCN, UNC-79*, UNC-80* and FAM155A* constructs ( and ) were generated using the Q5 Site-Directed Mutagenesis Kit. .. The sequences of purified plasmid DNAs from transformed E. coli were verified by Sanger DNA sequencing (Eurofins Genonimcs). .. For expression in Xenopus laevis oocytes, plasmid DNAs were linearized with XbaI restriction enzyme, from which capped RNAs were synthesised using the T7 mMessage mMachine Kit (Ambion).

Synthesized:

Article Title: C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation
Article Snippet: All chemicals used for ORi preparation were purchased from Fluka (Deisenhofen, Germany), except for HEPES, penicillin, and streptomycin (Sigma-Aldrich). .. Plasmid DNAs encoding the human CHRNA9 and CHRNA10 as well as the 43 kDa receptor-associated protein of the synapse ( RAPSN ) were obtained from Eurofins Genomics (Ebersberg, Germany) and capped cRNA was synthesized as described before ( ). .. Human CHRNA7 encoding cRNA was kindly provided by G. Schmalzing (Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany) and synthesized as described before ( ). cRNA was dissolved in nuclease-free water and injected into oocytes in a volume of 50.6 nl using a microinjector (Nanoject, Drummond Scientific, Broomall, PA, USA).

Clone Assay:

Article Title: Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA
Article Snippet: After heating to 95 °C (to switch from the RT reaction to the PCR, following the manufacturer’s instructions), the reverse primer was added (5′-GTCATTCTCCTAAGAAGC-3′). .. Cloning of the PCR products was performed with the CloneJET PCR Cloning Kit (Thermo Fisher Scientific), and plasmid DNAs were sequenced using a third primer (5′-CGATGAGTTTTCGGTATTATC-3′) by Eurofins Genomics. ..

Polymerase Chain Reaction:

Article Title: Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA
Article Snippet: After heating to 95 °C (to switch from the RT reaction to the PCR, following the manufacturer’s instructions), the reverse primer was added (5′-GTCATTCTCCTAAGAAGC-3′). .. Cloning of the PCR products was performed with the CloneJET PCR Cloning Kit (Thermo Fisher Scientific), and plasmid DNAs were sequenced using a third primer (5′-CGATGAGTTTTCGGTATTATC-3′) by Eurofins Genomics. ..

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    Eurofins plasmid dna dna oligonucleotides
    Effect of the GAS5 HREM <t>DNA</t> oligonucleotide on basal and UV-C-induced cell death after silencing of endogenous GAS5 expression in MCF7 cells Cells (n = 4 cultures) were transfected with either GAS5 siRNA (GAS5#4; targets exon 12 sequence) or negative control (NC) siRNA and, after 24 h, transfected with either the GAS5 HREM DNA oligonucleotide (HREM) or stem loop (SL) control DNA oligonucleotides. After 20 h, cells were irradiated with UV-C light, then plated for assessment of cell survival after a further 48 h. The GAS5#4 siRNA markedly reduced GAS5 lncRNA levels at 24 h post -transfection (panel A ) i.e. , immediately prior to <t>nucleofection</t> of DNA oligonucleotides. The HREM oligonucleotide induced apoptosis (panel B ) and decreased culture viability (panel C ) at 20 h post -transfection, irrespective of endogenous GAS5 lncRNA levels; a similar pattern was found for apoptosis (panel D ) and culture viability (panel E ) in mock-irradiated controls ( i.e. at 68 h post -oligonucleotide transfection). Silencing of GAS5 attenuated UV-C induced apoptosis (panel F ) and the associated loss of culture viability (panel G ) at 48 h post -irradiation, but had no effect cell death induction by the HREM oligonucleotide. Panel A: *** P
    Plasmid Dna Dna Oligonucleotides, supplied by Eurofins, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid dna dna oligonucleotides/product/Eurofins
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid dna dna oligonucleotides - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    86
    Eurofins plasmid dna
    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. <t>DNA</t> was sequenced using <t>M13</t> primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).
    Plasmid Dna, supplied by Eurofins, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of the GAS5 HREM DNA oligonucleotide on basal and UV-C-induced cell death after silencing of endogenous GAS5 expression in MCF7 cells Cells (n = 4 cultures) were transfected with either GAS5 siRNA (GAS5#4; targets exon 12 sequence) or negative control (NC) siRNA and, after 24 h, transfected with either the GAS5 HREM DNA oligonucleotide (HREM) or stem loop (SL) control DNA oligonucleotides. After 20 h, cells were irradiated with UV-C light, then plated for assessment of cell survival after a further 48 h. The GAS5#4 siRNA markedly reduced GAS5 lncRNA levels at 24 h post -transfection (panel A ) i.e. , immediately prior to nucleofection of DNA oligonucleotides. The HREM oligonucleotide induced apoptosis (panel B ) and decreased culture viability (panel C ) at 20 h post -transfection, irrespective of endogenous GAS5 lncRNA levels; a similar pattern was found for apoptosis (panel D ) and culture viability (panel E ) in mock-irradiated controls ( i.e. at 68 h post -oligonucleotide transfection). Silencing of GAS5 attenuated UV-C induced apoptosis (panel F ) and the associated loss of culture viability (panel G ) at 48 h post -irradiation, but had no effect cell death induction by the HREM oligonucleotide. Panel A: *** P

    Journal: Oncotarget

    Article Title: The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells

    doi: 10.18632/oncotarget.7173

    Figure Lengend Snippet: Effect of the GAS5 HREM DNA oligonucleotide on basal and UV-C-induced cell death after silencing of endogenous GAS5 expression in MCF7 cells Cells (n = 4 cultures) were transfected with either GAS5 siRNA (GAS5#4; targets exon 12 sequence) or negative control (NC) siRNA and, after 24 h, transfected with either the GAS5 HREM DNA oligonucleotide (HREM) or stem loop (SL) control DNA oligonucleotides. After 20 h, cells were irradiated with UV-C light, then plated for assessment of cell survival after a further 48 h. The GAS5#4 siRNA markedly reduced GAS5 lncRNA levels at 24 h post -transfection (panel A ) i.e. , immediately prior to nucleofection of DNA oligonucleotides. The HREM oligonucleotide induced apoptosis (panel B ) and decreased culture viability (panel C ) at 20 h post -transfection, irrespective of endogenous GAS5 lncRNA levels; a similar pattern was found for apoptosis (panel D ) and culture viability (panel E ) in mock-irradiated controls ( i.e. at 68 h post -oligonucleotide transfection). Silencing of GAS5 attenuated UV-C induced apoptosis (panel F ) and the associated loss of culture viability (panel G ) at 48 h post -irradiation, but had no effect cell death induction by the HREM oligonucleotide. Panel A: *** P

    Article Snippet: Nucleofection of oligonucleotides and plasmid DNA DNA oligonucleotides (Eurofins Genomics, Ebersberg, Germany) were studied in the majority of experiments and comprised the wild-type GAS5 HREM sequence (5′-CAGTGGTCTTTGTAGACTGCCTG-3′), a mutated GAS5 HREM (5′-CAGTAGTCTTTGTAGACTGCCTG-3′), a control sequence which retains stem complementarity but lacks the GAS5 HRE consensus (stem loop control; 5′-CTGATGGTCTTTGTAGACCATCA-3′) and scrambled sequence (5′-TGTTGGCTTGTCACGCATGCGTCT-3′).

    Techniques: Expressing, Transfection, Sequencing, Negative Control, Irradiation

    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Journal: PLoS ONE

    Article Title: The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion

    doi: 10.1371/journal.pone.0205664

    Figure Lengend Snippet: The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Article Snippet: Subsequently, the plasmid DNA was isolated and sequenced using M13 primers ( 5'-TGT AAA ACG ACG GCC AGT-3' (forward), 5'-CAG GAA ACA GCT ATG ACC-3' (reverse)) by MWG eurofins, to ascertain where the transcription start site was on the sequence, as the inserted fragment sequence would begin at the transcription start site, preceded by the universal primer sequence.

    Techniques: Isolation, Sequencing, Activity Assay, In Silico, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Journal: PLoS ONE

    Article Title: The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion

    doi: 10.1371/journal.pone.0205664

    Figure Lengend Snippet: The MSTN transcription start site is altered by the SINE insertion. RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

    Article Snippet: Subsequently, the plasmid DNA was isolated and sequenced using M13 primers ( 5'-TGT AAA ACG ACG GCC AGT-3' (forward), 5'-CAG GAA ACA GCT ATG ACC-3' (reverse)) by MWG eurofins, to ascertain where the transcription start site was on the sequence, as the inserted fragment sequence would begin at the transcription start site, preceded by the universal primer sequence.

    Techniques: Isolation, Sequencing, Activity Assay, In Silico, Polymerase Chain Reaction, Agarose Gel Electrophoresis