Structured Review

Boehringer Mannheim plasmid dna
Complete nucleotide and amino acid sequences of <t>NucA.</t> The presumed ribosome binding site is labeled RBS. Amino acids are listed starting with the initiation codon, methionine. The signal sequence is underlined, and the start codon, lysine, of the mature sequence is shown by an arrow. The locations of various restriction sites are displayed above the sequence. The boxed region demarcates the region of strong protein homology observed among known 5′-nucleotidases. Arrows below the <t>DNA</t> sequence at the 3′ end of the gene depict an inverted-repeat region. ORF, open reading frame.
Plasmid Dna, supplied by Boehringer Mannheim, 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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Images

1) Product Images from "Identification of a Haemophilus influenzae 5?-Nucleotidase Protein: Cloning of the nucA Gene and Immunogenicity and Characterization of the NucA Protein"

Article Title: Identification of a Haemophilus influenzae 5?-Nucleotidase Protein: Cloning of the nucA Gene and Immunogenicity and Characterization of the NucA Protein

Journal: Infection and Immunity

doi:

Complete nucleotide and amino acid sequences of NucA. The presumed ribosome binding site is labeled RBS. Amino acids are listed starting with the initiation codon, methionine. The signal sequence is underlined, and the start codon, lysine, of the mature sequence is shown by an arrow. The locations of various restriction sites are displayed above the sequence. The boxed region demarcates the region of strong protein homology observed among known 5′-nucleotidases. Arrows below the DNA sequence at the 3′ end of the gene depict an inverted-repeat region. ORF, open reading frame.
Figure Legend Snippet: Complete nucleotide and amino acid sequences of NucA. The presumed ribosome binding site is labeled RBS. Amino acids are listed starting with the initiation codon, methionine. The signal sequence is underlined, and the start codon, lysine, of the mature sequence is shown by an arrow. The locations of various restriction sites are displayed above the sequence. The boxed region demarcates the region of strong protein homology observed among known 5′-nucleotidases. Arrows below the DNA sequence at the 3′ end of the gene depict an inverted-repeat region. ORF, open reading frame.

Techniques Used: Binding Assay, Labeling, Sequencing

2) Product Images from "PTTG/securin activates expression of p53 and modulates its function"

Article Title: PTTG/securin activates expression of p53 and modulates its function

Journal: Molecular Cancer

doi: 10.1186/1476-4598-3-18

Electrophoretic mobility shift assays show the binding of c-myc protein to the c-myc/max sequence. A : Nuclear extract prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 gene promoter sequence carrying a normal c-myc/max binding site. Addition of a 20-fold molar excess of specific unlabeled DNA resulted in almost complete disappearance of the DNA-protein complex (lane 3). An arrow indicates the specific DNA-protein complex. B : Nuclear extracts prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 promoter sequence carrying a normal c-myc/max binding site. Addition of antibody directed against the N-terminal of c-myc resulted in a supershift (lane 3, indicated by an arrow) whereas no supershift was obtained when the [ 32 P]-labeled-D-172/-89 c-myc sequence was used as a probe (lane 5). Addition of antibodies directed against the C-terminal of c-myc did not result in supershift (lanes 4 6) when either of the probes was used in the binding reaction indicating that C-terminal of c-myc is not assessable. N.S indicates non-specific complex.
Figure Legend Snippet: Electrophoretic mobility shift assays show the binding of c-myc protein to the c-myc/max sequence. A : Nuclear extract prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 gene promoter sequence carrying a normal c-myc/max binding site. Addition of a 20-fold molar excess of specific unlabeled DNA resulted in almost complete disappearance of the DNA-protein complex (lane 3). An arrow indicates the specific DNA-protein complex. B : Nuclear extracts prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 promoter sequence carrying a normal c-myc/max binding site. Addition of antibody directed against the N-terminal of c-myc resulted in a supershift (lane 3, indicated by an arrow) whereas no supershift was obtained when the [ 32 P]-labeled-D-172/-89 c-myc sequence was used as a probe (lane 5). Addition of antibodies directed against the C-terminal of c-myc did not result in supershift (lanes 4 6) when either of the probes was used in the binding reaction indicating that C-terminal of c-myc is not assessable. N.S indicates non-specific complex.

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Transfection, Labeling

3) Product Images from "Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx"

Article Title: Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx

Journal: Journal of Virology

doi: 10.1128/JVI.76.11.5769-5783.2002

Delineation of the pp71 interaction domain within hDaxx. (A, B, and C) Yeast cells were transformed with two separate vectors, one of which encoded either PML as a positive control (A) or pp71 (C) fused to the GAL4 DNA-binding domain. The second plasmid encoded amino- or carboxy-terminal fragments of hDaxx as fusion with the GAL4 activation domain; the amino acids contained in the respective deletion mutants are indicated in panel B. Yeast colonies were selected for the presence of both plasmids on dropout medium lacking tryptophane and leucine and subsequently analyzed for the expression of β-galactosidase by filter lift assays. As negative controls, the activation domain vector pGAD424 (pGAD) was either transformed with the pp71 or the PML DNA-binding domain fusion (lanes 11, panels A and C, respectively). The pp71 interaction domain within hDaxx is depicted by the box. (D) Interaction between pp71 and hDaxx deletion mutants after coimmunoprecipitation from 293 cells. The hDaxx mutants were precipitated with the anti-FLAG monoclonal antibody; therafter, bound pp71 protein was detected in Western blot experiments employing the anti-myc antibody. Lanes: 1, lysates from untransfected cells; 2, transfection with plasmid myc-pp71 alone; 3, transfection with vector FLAG-hDaxx alone; 4, transfection with a combination of vectors encoding myc-pp71 and FLAG-hDaxx; 5, transfection with a plasmid encoding FLAG-hDaxx 43-501; 6, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 43-501; 7, transfection with vector FLAG-hDaxx 43-371; 8, transfection with a combination of vectors myc-pp71 and FLAG-hDaxx 43-371; 9, transfection with a plasmid expressing FLAG-hDaxx 197-439; 10, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 197-439; 11, transfection with plasmid FLAG-hDaxx 371-740; 12, transfection with a combination of vector myc-pp71 and FLAG-hDaxx 371-740; 13, transfection with a plasmid encoding FLAG-hDaxx 538-740; 14, transfection with vectors expressing myc-pp71 and FLAG-hDaxx 538-740. (E) Western blot analysis of the cell lysates used for immunoprecipitation in panel D. The expression of the hDaxx deletion mutants was investigated with the anti-FLAG monoclonal antibody. Molecular masses are indicated in kilodaltons. Abbreviations: IP, immunoprecipitation; wb, Western blot; IgG, immunoglobulin G.
Figure Legend Snippet: Delineation of the pp71 interaction domain within hDaxx. (A, B, and C) Yeast cells were transformed with two separate vectors, one of which encoded either PML as a positive control (A) or pp71 (C) fused to the GAL4 DNA-binding domain. The second plasmid encoded amino- or carboxy-terminal fragments of hDaxx as fusion with the GAL4 activation domain; the amino acids contained in the respective deletion mutants are indicated in panel B. Yeast colonies were selected for the presence of both plasmids on dropout medium lacking tryptophane and leucine and subsequently analyzed for the expression of β-galactosidase by filter lift assays. As negative controls, the activation domain vector pGAD424 (pGAD) was either transformed with the pp71 or the PML DNA-binding domain fusion (lanes 11, panels A and C, respectively). The pp71 interaction domain within hDaxx is depicted by the box. (D) Interaction between pp71 and hDaxx deletion mutants after coimmunoprecipitation from 293 cells. The hDaxx mutants were precipitated with the anti-FLAG monoclonal antibody; therafter, bound pp71 protein was detected in Western blot experiments employing the anti-myc antibody. Lanes: 1, lysates from untransfected cells; 2, transfection with plasmid myc-pp71 alone; 3, transfection with vector FLAG-hDaxx alone; 4, transfection with a combination of vectors encoding myc-pp71 and FLAG-hDaxx; 5, transfection with a plasmid encoding FLAG-hDaxx 43-501; 6, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 43-501; 7, transfection with vector FLAG-hDaxx 43-371; 8, transfection with a combination of vectors myc-pp71 and FLAG-hDaxx 43-371; 9, transfection with a plasmid expressing FLAG-hDaxx 197-439; 10, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 197-439; 11, transfection with plasmid FLAG-hDaxx 371-740; 12, transfection with a combination of vector myc-pp71 and FLAG-hDaxx 371-740; 13, transfection with a plasmid encoding FLAG-hDaxx 538-740; 14, transfection with vectors expressing myc-pp71 and FLAG-hDaxx 538-740. (E) Western blot analysis of the cell lysates used for immunoprecipitation in panel D. The expression of the hDaxx deletion mutants was investigated with the anti-FLAG monoclonal antibody. Molecular masses are indicated in kilodaltons. Abbreviations: IP, immunoprecipitation; wb, Western blot; IgG, immunoglobulin G.

Techniques Used: Transformation Assay, Positive Control, Binding Assay, Plasmid Preparation, Activation Assay, Expressing, Western Blot, Transfection, Immunoprecipitation

4) Product Images from "Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo"

Article Title: Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

Journal: Molecular and Cellular Biology

doi:

Immunoblot detection of PTB in transfected BS-C-1 and Huh-7 cells in comparison to increases in translation directed by the wt IRES of HAV. (A) Relative expression of renilla luciferase (RLuc) following DNA transfection of cells with a dicistronic plasmid expressing wt PTB from the upstream cistron, compared with a matched control plasmid encoding the PTB null mutant, Δ87-531, in the upstream cistron. RLuc was translated from the downstream cistron by a cap-independent process under control of the wt 5′NTR of HAV. Transfection was by a liposome-mediated procedure. (B) Immunoblot analysis of BS-C-1 and Huh-7 cells following DNA transfection under conditions identical to those used in panel A with monocistronic plasmids expressing either wt PTB or the null mutant, Δ87-531. C, cytoplasmic fraction; N, nuclear fraction.
Figure Legend Snippet: Immunoblot detection of PTB in transfected BS-C-1 and Huh-7 cells in comparison to increases in translation directed by the wt IRES of HAV. (A) Relative expression of renilla luciferase (RLuc) following DNA transfection of cells with a dicistronic plasmid expressing wt PTB from the upstream cistron, compared with a matched control plasmid encoding the PTB null mutant, Δ87-531, in the upstream cistron. RLuc was translated from the downstream cistron by a cap-independent process under control of the wt 5′NTR of HAV. Transfection was by a liposome-mediated procedure. (B) Immunoblot analysis of BS-C-1 and Huh-7 cells following DNA transfection under conditions identical to those used in panel A with monocistronic plasmids expressing either wt PTB or the null mutant, Δ87-531. C, cytoplasmic fraction; N, nuclear fraction.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Mutagenesis

Northern blot analysis of the poly(A) fraction of RNA extracted from BS-C-1 cells following DNA transfection with constructs containing the HAV IRES, pPwt/AC (lane 1), or its related null mutant pΔ87-531/AC (lane 2). Lane 3 was loaded with RNA from mock-transfected cells. The probe for hybridization was complementary to the CAT sequence.
Figure Legend Snippet: Northern blot analysis of the poly(A) fraction of RNA extracted from BS-C-1 cells following DNA transfection with constructs containing the HAV IRES, pPwt/AC (lane 1), or its related null mutant pΔ87-531/AC (lane 2). Lane 3 was loaded with RNA from mock-transfected cells. The probe for hybridization was complementary to the CAT sequence.

Techniques Used: Northern Blot, Transfection, Construct, Mutagenesis, Hybridization, Sequencing

5) Product Images from "Pax8 has a key role in thyroid cell differentiation"

Article Title: Pax8 has a key role in thyroid cell differentiation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Analysis of the transcriptional properties of Pax8 splicing isoforms. ( A ) Northern blot analysis of total RNA prepared from PCPy cells stably transfected with Pax8 splicing isoforms. Tg expression was analyzed in two different pools of clones for each transfected DNA. The GAPDH hybridization was used to normalize the amount of RNA present in each lane. ( B ) Reverse transcription–PCR analysis of cDNA derived from total RNA from the same cells as in A . Pax8 expression was analyzed in two different pools of clones for each transfected DNA.
Figure Legend Snippet: Analysis of the transcriptional properties of Pax8 splicing isoforms. ( A ) Northern blot analysis of total RNA prepared from PCPy cells stably transfected with Pax8 splicing isoforms. Tg expression was analyzed in two different pools of clones for each transfected DNA. The GAPDH hybridization was used to normalize the amount of RNA present in each lane. ( B ) Reverse transcription–PCR analysis of cDNA derived from total RNA from the same cells as in A . Pax8 expression was analyzed in two different pools of clones for each transfected DNA.

Techniques Used: Northern Blot, Stable Transfection, Transfection, Expressing, Clone Assay, Hybridization, Polymerase Chain Reaction, Derivative Assay

Related Articles

Generated:

Article Title: Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp ( Penaeus monodon)
Article Snippet: A GEM-T Easy plasmid containing a region (1856–2175 nt) of P monodon hsc70 cDNA was used as a template for the preparation of the probes. .. Digoxigenin (DIG)-uridine triphosphate–labeled sense and antisense riboprobes were generated from linearized cDNA plasmids (5 μg) by in vitro transcription using the RNA-labeling kits T7 RNA polymerase and SP6 RNA polymerase (Boehringer Mannheim), respectively. ..

In Vitro:

Article Title: Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp ( Penaeus monodon)
Article Snippet: A GEM-T Easy plasmid containing a region (1856–2175 nt) of P monodon hsc70 cDNA was used as a template for the preparation of the probes. .. Digoxigenin (DIG)-uridine triphosphate–labeled sense and antisense riboprobes were generated from linearized cDNA plasmids (5 μg) by in vitro transcription using the RNA-labeling kits T7 RNA polymerase and SP6 RNA polymerase (Boehringer Mannheim), respectively. ..

Plasmid Preparation:

Article Title: vanA Gene Cluster in a Vancomycin-Resistant Clinical Isolate of Bacillus circulans
Article Snippet: The PCR mixtures were denatured (2 min at 94°C) and were then subjected to 35 cycles of amplification (1 min of annealing at 55°C, 1 min of elongation at 72°C, and 1 min of denaturation at 94°C) and a final elongation step of 72°C for 10 min. PCR products were resolved by electrophoresis in a 1% agarose gel stained with ethidium bromide. .. Genomic and plasmid DNAs were digested with restriction enzymes as recommended by the manufacturer (Boehringer Mannheim) and were electrophoresed through a 0.8% agarose gel. .. The DNA restriction fragments were electroblotted onto a Hybond-N+ nylon membrane (Amersham International, Amersham, United Kingdom) and were hybridized with the DIG-dUTP-labeled PCR products.

Article Title: Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors †
Article Snippet: The Escherichia coli β-galactosidase (β-Gal) gene, which was PCR amplified from the C20DX/β-gal/2Arep construct ( ) by using NsiLacZ_F and NsiLacZ_R primers with incorporated Nsi I restriction sites (Table ), was cloned as a reporter gene into the Nsi I sites of the pKUNrep2, pKUNrep2(dGDD), pKUNrep3, and pKUNrep4 vectors, producing the pKUNβrep2, pKUNβrep2(dGDD), pKUNβrep3, and pKUNβrep4 constructs, respectively. .. Plasmid DNAs were transfected with FuGENE 6 transfection reagent (Boehringer Mannheim) essentially as described by the manufacturer. .. For HG expression and stable cell line selection, ∼0.8 μg of DNA was used with 2 μl of FuGENE 6 to transfect ∼1.3 × 105 cells in 16-mm-diameter wells (of a 24-well cell culture plate).

Article Title: Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
Article Snippet: The cells were labeled for 4 h with 25 Ci/ml [ 35 S]methionine-cysteine at 6 h postinfection before harvesting. .. Radiolabeling of IBV-infected and mock-infected Vero cells Transient expression of plasmid DNAs using the vaccinia virus-T7 expression system Semiconfluent monolayers of Cos-7 cells grown on 60-mm dishes were infected with 10 PFU/cell of vTF7-3 (Fuerst et al., 1986) and transfected with 2.5 g of plasmid DNAs using 15 l of DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation of the cells at 37°C for 7 h, the cells were incubated in methionine-and cysteine-free medium (ICN) for 30 min and labeled with 25 Ci/ml [ 35 S]methionine-cysteine (35S Express Protein Labelling Mix, NEN Life Science) for 13 h. The cells were scraped off the dishes in phosphate-buffered saline (PBS) and recovered by centrifugation at 12,000 rpm for 1 min.

Article Title: The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection.
Article Snippet: The SpeI (blunt-ended) EcoRI fragment of the BCV HE gene was cloned into the HindIII (blunt-ended) EcoRI sites of the pcDNA3 vector, resulting in pcDNA3/BHE. .. DNA transfection and selection of stable transfectants DBT cells were grown in MEM in 60-mm culture plates (Sarsted) to 60-70% confluence and were transfected with plasmid DNAs containing the pcDNA3 vector alone or vectors with the BCV S gene (pcDNA3/BS) or the HE gene (pcDNA3/BHE) by use of the cationic liposome transfection reagent DOTAP according to the manufacturer's instructions (Boehringer Mannheim). .. Briefly, 7 g of each construct was mixed with 14 g of DOTAP in 65 l of 20 mM HEPES (pH 7.4).

Article Title: Sequence of the FRA3B common fragile region: Implications for the mechanism of FHIT deletion
Article Snippet: Recombinant plasmids were isolated by Qiagen BioRobot 9600, and DNA concentration measured on the FluorImager SI analyzer. .. Plasmid DNAs were digested with Eco RI and Hin dIII (Boehringer Mannheim) and were gel separated to confirm the presence of insert DNA. .. Sequencing reactions and analysis were performed by using dyedeoxy-terminator reaction chemistry on a Perkin–Elmer/Cetus DNA Thermal Cycler 9600 and the Applied Biosystems Model 377 DNA sequencing systems.

Agarose Gel Electrophoresis:

Article Title: vanA Gene Cluster in a Vancomycin-Resistant Clinical Isolate of Bacillus circulans
Article Snippet: The PCR mixtures were denatured (2 min at 94°C) and were then subjected to 35 cycles of amplification (1 min of annealing at 55°C, 1 min of elongation at 72°C, and 1 min of denaturation at 94°C) and a final elongation step of 72°C for 10 min. PCR products were resolved by electrophoresis in a 1% agarose gel stained with ethidium bromide. .. Genomic and plasmid DNAs were digested with restriction enzymes as recommended by the manufacturer (Boehringer Mannheim) and were electrophoresed through a 0.8% agarose gel. .. The DNA restriction fragments were electroblotted onto a Hybond-N+ nylon membrane (Amersham International, Amersham, United Kingdom) and were hybridized with the DIG-dUTP-labeled PCR products.

Transfection:

Article Title: Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors †
Article Snippet: The Escherichia coli β-galactosidase (β-Gal) gene, which was PCR amplified from the C20DX/β-gal/2Arep construct ( ) by using NsiLacZ_F and NsiLacZ_R primers with incorporated Nsi I restriction sites (Table ), was cloned as a reporter gene into the Nsi I sites of the pKUNrep2, pKUNrep2(dGDD), pKUNrep3, and pKUNrep4 vectors, producing the pKUNβrep2, pKUNβrep2(dGDD), pKUNβrep3, and pKUNβrep4 constructs, respectively. .. Plasmid DNAs were transfected with FuGENE 6 transfection reagent (Boehringer Mannheim) essentially as described by the manufacturer. .. For HG expression and stable cell line selection, ∼0.8 μg of DNA was used with 2 μl of FuGENE 6 to transfect ∼1.3 × 105 cells in 16-mm-diameter wells (of a 24-well cell culture plate).

Article Title: Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
Article Snippet: The cells were labeled for 4 h with 25 Ci/ml [ 35 S]methionine-cysteine at 6 h postinfection before harvesting. .. Radiolabeling of IBV-infected and mock-infected Vero cells Transient expression of plasmid DNAs using the vaccinia virus-T7 expression system Semiconfluent monolayers of Cos-7 cells grown on 60-mm dishes were infected with 10 PFU/cell of vTF7-3 (Fuerst et al., 1986) and transfected with 2.5 g of plasmid DNAs using 15 l of DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation of the cells at 37°C for 7 h, the cells were incubated in methionine-and cysteine-free medium (ICN) for 30 min and labeled with 25 Ci/ml [ 35 S]methionine-cysteine (35S Express Protein Labelling Mix, NEN Life Science) for 13 h. The cells were scraped off the dishes in phosphate-buffered saline (PBS) and recovered by centrifugation at 12,000 rpm for 1 min.

Article Title: The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection.
Article Snippet: The SpeI (blunt-ended) EcoRI fragment of the BCV HE gene was cloned into the HindIII (blunt-ended) EcoRI sites of the pcDNA3 vector, resulting in pcDNA3/BHE. .. DNA transfection and selection of stable transfectants DBT cells were grown in MEM in 60-mm culture plates (Sarsted) to 60-70% confluence and were transfected with plasmid DNAs containing the pcDNA3 vector alone or vectors with the BCV S gene (pcDNA3/BS) or the HE gene (pcDNA3/BHE) by use of the cationic liposome transfection reagent DOTAP according to the manufacturer's instructions (Boehringer Mannheim). .. Briefly, 7 g of each construct was mixed with 14 g of DOTAP in 65 l of 20 mM HEPES (pH 7.4).

Radioactivity:

Article Title: Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
Article Snippet: The cells were labeled for 4 h with 25 Ci/ml [ 35 S]methionine-cysteine at 6 h postinfection before harvesting. .. Radiolabeling of IBV-infected and mock-infected Vero cells Transient expression of plasmid DNAs using the vaccinia virus-T7 expression system Semiconfluent monolayers of Cos-7 cells grown on 60-mm dishes were infected with 10 PFU/cell of vTF7-3 (Fuerst et al., 1986) and transfected with 2.5 g of plasmid DNAs using 15 l of DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation of the cells at 37°C for 7 h, the cells were incubated in methionine-and cysteine-free medium (ICN) for 30 min and labeled with 25 Ci/ml [ 35 S]methionine-cysteine (35S Express Protein Labelling Mix, NEN Life Science) for 13 h. The cells were scraped off the dishes in phosphate-buffered saline (PBS) and recovered by centrifugation at 12,000 rpm for 1 min.

Expressing:

Article Title: Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
Article Snippet: The cells were labeled for 4 h with 25 Ci/ml [ 35 S]methionine-cysteine at 6 h postinfection before harvesting. .. Radiolabeling of IBV-infected and mock-infected Vero cells Transient expression of plasmid DNAs using the vaccinia virus-T7 expression system Semiconfluent monolayers of Cos-7 cells grown on 60-mm dishes were infected with 10 PFU/cell of vTF7-3 (Fuerst et al., 1986) and transfected with 2.5 g of plasmid DNAs using 15 l of DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation of the cells at 37°C for 7 h, the cells were incubated in methionine-and cysteine-free medium (ICN) for 30 min and labeled with 25 Ci/ml [ 35 S]methionine-cysteine (35S Express Protein Labelling Mix, NEN Life Science) for 13 h. The cells were scraped off the dishes in phosphate-buffered saline (PBS) and recovered by centrifugation at 12,000 rpm for 1 min.

Infection:

Article Title: Further characterization of the coronavirus infectious bronchitis virus 3C-like proteinase and determination of a new cleavage site.
Article Snippet: The cells were labeled for 4 h with 25 Ci/ml [ 35 S]methionine-cysteine at 6 h postinfection before harvesting. .. Radiolabeling of IBV-infected and mock-infected Vero cells Transient expression of plasmid DNAs using the vaccinia virus-T7 expression system Semiconfluent monolayers of Cos-7 cells grown on 60-mm dishes were infected with 10 PFU/cell of vTF7-3 (Fuerst et al., 1986) and transfected with 2.5 g of plasmid DNAs using 15 l of DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. After incubation of the cells at 37°C for 7 h, the cells were incubated in methionine-and cysteine-free medium (ICN) for 30 min and labeled with 25 Ci/ml [ 35 S]methionine-cysteine (35S Express Protein Labelling Mix, NEN Life Science) for 13 h. The cells were scraped off the dishes in phosphate-buffered saline (PBS) and recovered by centrifugation at 12,000 rpm for 1 min.

Selection:

Article Title: The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection.
Article Snippet: The SpeI (blunt-ended) EcoRI fragment of the BCV HE gene was cloned into the HindIII (blunt-ended) EcoRI sites of the pcDNA3 vector, resulting in pcDNA3/BHE. .. DNA transfection and selection of stable transfectants DBT cells were grown in MEM in 60-mm culture plates (Sarsted) to 60-70% confluence and were transfected with plasmid DNAs containing the pcDNA3 vector alone or vectors with the BCV S gene (pcDNA3/BS) or the HE gene (pcDNA3/BHE) by use of the cationic liposome transfection reagent DOTAP according to the manufacturer's instructions (Boehringer Mannheim). .. Briefly, 7 g of each construct was mixed with 14 g of DOTAP in 65 l of 20 mM HEPES (pH 7.4).

Cell Culture:

Article Title: DNA Vaccination with the Hantaan Virus M Gene Protects Hamsters against Three of Four HFRS Hantaviruses and Elicits a High-Titer Neutralizing Antibody Response in Rhesus Monkeys
Article Snippet: Plasmid DNA was purified by using Qiagen Maxiprep DNA purification kits according to the manufacturer's directions. .. COS cells grown in T-25 cell culture flasks were transfected with 5 μg of plasmid DNA with Fugene6 (Boehringer Mannheim). .. After 24 h, expression products were radiolabeled with Promix ([35 S]methionine and [35 S]cysteine; Amersham) and immunoprecipitated as described previously ( ).

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    Boehringer Mannheim plasmid dna
    Delineation of the pp71 interaction domain within hDaxx. (A, B, and C) Yeast cells were transformed with two separate vectors, one of which encoded either PML as a positive control (A) or pp71 (C) fused to the GAL4 <t>DNA-binding</t> domain. The second plasmid encoded amino- or carboxy-terminal fragments of hDaxx as fusion with the GAL4 activation domain; the amino acids contained in the respective deletion mutants are indicated in panel B. Yeast colonies were selected for the presence of both plasmids on dropout medium lacking tryptophane and leucine and subsequently analyzed for the expression of β-galactosidase by filter lift assays. As negative controls, the activation domain vector pGAD424 (pGAD) was either transformed with the pp71 or the PML DNA-binding domain fusion (lanes 11, panels A and C, respectively). The pp71 interaction domain within hDaxx is depicted by the box. (D) Interaction between pp71 and hDaxx deletion mutants after coimmunoprecipitation from 293 cells. The hDaxx mutants were precipitated with the anti-FLAG monoclonal antibody; therafter, bound pp71 protein was detected in Western blot experiments employing the anti-myc antibody. Lanes: 1, lysates from untransfected cells; 2, <t>transfection</t> with plasmid myc-pp71 alone; 3, transfection with vector FLAG-hDaxx alone; 4, transfection with a combination of vectors encoding myc-pp71 and FLAG-hDaxx; 5, transfection with a plasmid encoding FLAG-hDaxx 43-501; 6, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 43-501; 7, transfection with vector FLAG-hDaxx 43-371; 8, transfection with a combination of vectors myc-pp71 and FLAG-hDaxx 43-371; 9, transfection with a plasmid expressing FLAG-hDaxx 197-439; 10, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 197-439; 11, transfection with plasmid FLAG-hDaxx 371-740; 12, transfection with a combination of vector myc-pp71 and FLAG-hDaxx 371-740; 13, transfection with a plasmid encoding FLAG-hDaxx 538-740; 14, transfection with vectors expressing myc-pp71 and FLAG-hDaxx 538-740. (E) Western blot analysis of the cell lysates used for immunoprecipitation in panel D. The expression of the hDaxx deletion mutants was investigated with the anti-FLAG monoclonal antibody. Molecular masses are indicated in kilodaltons. Abbreviations: IP, immunoprecipitation; wb, Western blot; IgG, immunoglobulin G.
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    Delineation of the pp71 interaction domain within hDaxx. (A, B, and C) Yeast cells were transformed with two separate vectors, one of which encoded either PML as a positive control (A) or pp71 (C) fused to the GAL4 DNA-binding domain. The second plasmid encoded amino- or carboxy-terminal fragments of hDaxx as fusion with the GAL4 activation domain; the amino acids contained in the respective deletion mutants are indicated in panel B. Yeast colonies were selected for the presence of both plasmids on dropout medium lacking tryptophane and leucine and subsequently analyzed for the expression of β-galactosidase by filter lift assays. As negative controls, the activation domain vector pGAD424 (pGAD) was either transformed with the pp71 or the PML DNA-binding domain fusion (lanes 11, panels A and C, respectively). The pp71 interaction domain within hDaxx is depicted by the box. (D) Interaction between pp71 and hDaxx deletion mutants after coimmunoprecipitation from 293 cells. The hDaxx mutants were precipitated with the anti-FLAG monoclonal antibody; therafter, bound pp71 protein was detected in Western blot experiments employing the anti-myc antibody. Lanes: 1, lysates from untransfected cells; 2, transfection with plasmid myc-pp71 alone; 3, transfection with vector FLAG-hDaxx alone; 4, transfection with a combination of vectors encoding myc-pp71 and FLAG-hDaxx; 5, transfection with a plasmid encoding FLAG-hDaxx 43-501; 6, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 43-501; 7, transfection with vector FLAG-hDaxx 43-371; 8, transfection with a combination of vectors myc-pp71 and FLAG-hDaxx 43-371; 9, transfection with a plasmid expressing FLAG-hDaxx 197-439; 10, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 197-439; 11, transfection with plasmid FLAG-hDaxx 371-740; 12, transfection with a combination of vector myc-pp71 and FLAG-hDaxx 371-740; 13, transfection with a plasmid encoding FLAG-hDaxx 538-740; 14, transfection with vectors expressing myc-pp71 and FLAG-hDaxx 538-740. (E) Western blot analysis of the cell lysates used for immunoprecipitation in panel D. The expression of the hDaxx deletion mutants was investigated with the anti-FLAG monoclonal antibody. Molecular masses are indicated in kilodaltons. Abbreviations: IP, immunoprecipitation; wb, Western blot; IgG, immunoglobulin G.

    Journal: Journal of Virology

    Article Title: Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx

    doi: 10.1128/JVI.76.11.5769-5783.2002

    Figure Lengend Snippet: Delineation of the pp71 interaction domain within hDaxx. (A, B, and C) Yeast cells were transformed with two separate vectors, one of which encoded either PML as a positive control (A) or pp71 (C) fused to the GAL4 DNA-binding domain. The second plasmid encoded amino- or carboxy-terminal fragments of hDaxx as fusion with the GAL4 activation domain; the amino acids contained in the respective deletion mutants are indicated in panel B. Yeast colonies were selected for the presence of both plasmids on dropout medium lacking tryptophane and leucine and subsequently analyzed for the expression of β-galactosidase by filter lift assays. As negative controls, the activation domain vector pGAD424 (pGAD) was either transformed with the pp71 or the PML DNA-binding domain fusion (lanes 11, panels A and C, respectively). The pp71 interaction domain within hDaxx is depicted by the box. (D) Interaction between pp71 and hDaxx deletion mutants after coimmunoprecipitation from 293 cells. The hDaxx mutants were precipitated with the anti-FLAG monoclonal antibody; therafter, bound pp71 protein was detected in Western blot experiments employing the anti-myc antibody. Lanes: 1, lysates from untransfected cells; 2, transfection with plasmid myc-pp71 alone; 3, transfection with vector FLAG-hDaxx alone; 4, transfection with a combination of vectors encoding myc-pp71 and FLAG-hDaxx; 5, transfection with a plasmid encoding FLAG-hDaxx 43-501; 6, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 43-501; 7, transfection with vector FLAG-hDaxx 43-371; 8, transfection with a combination of vectors myc-pp71 and FLAG-hDaxx 43-371; 9, transfection with a plasmid expressing FLAG-hDaxx 197-439; 10, transfection with vectors encoding myc-pp71 and FLAG-hDaxx 197-439; 11, transfection with plasmid FLAG-hDaxx 371-740; 12, transfection with a combination of vector myc-pp71 and FLAG-hDaxx 371-740; 13, transfection with a plasmid encoding FLAG-hDaxx 538-740; 14, transfection with vectors expressing myc-pp71 and FLAG-hDaxx 538-740. (E) Western blot analysis of the cell lysates used for immunoprecipitation in panel D. The expression of the hDaxx deletion mutants was investigated with the anti-FLAG monoclonal antibody. Molecular masses are indicated in kilodaltons. Abbreviations: IP, immunoprecipitation; wb, Western blot; IgG, immunoglobulin G.

    Article Snippet: For indirect immunofluorescence analysis, HFF cells grown on coverslips were transfected with 2 μg of plasmid DNA by using the FuGENE transfection reagent according to the manufacturer's protocol (Boehringer Mannheim, Mannheim, Germany).

    Techniques: Transformation Assay, Positive Control, Binding Assay, Plasmid Preparation, Activation Assay, Expressing, Western Blot, Transfection, Immunoprecipitation

    Immunoblot detection of PTB in transfected BS-C-1 and Huh-7 cells in comparison to increases in translation directed by the wt IRES of HAV. (A) Relative expression of renilla luciferase (RLuc) following DNA transfection of cells with a dicistronic plasmid expressing wt PTB from the upstream cistron, compared with a matched control plasmid encoding the PTB null mutant, Δ87-531, in the upstream cistron. RLuc was translated from the downstream cistron by a cap-independent process under control of the wt 5′NTR of HAV. Transfection was by a liposome-mediated procedure. (B) Immunoblot analysis of BS-C-1 and Huh-7 cells following DNA transfection under conditions identical to those used in panel A with monocistronic plasmids expressing either wt PTB or the null mutant, Δ87-531. C, cytoplasmic fraction; N, nuclear fraction.

    Journal: Molecular and Cellular Biology

    Article Title: Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

    doi:

    Figure Lengend Snippet: Immunoblot detection of PTB in transfected BS-C-1 and Huh-7 cells in comparison to increases in translation directed by the wt IRES of HAV. (A) Relative expression of renilla luciferase (RLuc) following DNA transfection of cells with a dicistronic plasmid expressing wt PTB from the upstream cistron, compared with a matched control plasmid encoding the PTB null mutant, Δ87-531, in the upstream cistron. RLuc was translated from the downstream cistron by a cap-independent process under control of the wt 5′NTR of HAV. Transfection was by a liposome-mediated procedure. (B) Immunoblot analysis of BS-C-1 and Huh-7 cells following DNA transfection under conditions identical to those used in panel A with monocistronic plasmids expressing either wt PTB or the null mutant, Δ87-531. C, cytoplasmic fraction; N, nuclear fraction.

    Article Snippet: For these transfections, nearly confluent cells grown in 60-mm-diameter plastic dishes were transfected with plasmid DNA mixed with FuGENE 6 (Boehringer Mannheim); 100 μl of OptiMEM (Gibco-BRL) and 6 μl of FuGENE reagent were incubated for 10 min at room temperature prior to the addition of plasmid DNA (2 μg).

    Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Mutagenesis

    Northern blot analysis of the poly(A) fraction of RNA extracted from BS-C-1 cells following DNA transfection with constructs containing the HAV IRES, pPwt/AC (lane 1), or its related null mutant pΔ87-531/AC (lane 2). Lane 3 was loaded with RNA from mock-transfected cells. The probe for hybridization was complementary to the CAT sequence.

    Journal: Molecular and Cellular Biology

    Article Title: Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

    doi:

    Figure Lengend Snippet: Northern blot analysis of the poly(A) fraction of RNA extracted from BS-C-1 cells following DNA transfection with constructs containing the HAV IRES, pPwt/AC (lane 1), or its related null mutant pΔ87-531/AC (lane 2). Lane 3 was loaded with RNA from mock-transfected cells. The probe for hybridization was complementary to the CAT sequence.

    Article Snippet: For these transfections, nearly confluent cells grown in 60-mm-diameter plastic dishes were transfected with plasmid DNA mixed with FuGENE 6 (Boehringer Mannheim); 100 μl of OptiMEM (Gibco-BRL) and 6 μl of FuGENE reagent were incubated for 10 min at room temperature prior to the addition of plasmid DNA (2 μg).

    Techniques: Northern Blot, Transfection, Construct, Mutagenesis, Hybridization, Sequencing

    Analysis of the transcriptional properties of Pax8 splicing isoforms. ( A ) Northern blot analysis of total RNA prepared from PCPy cells stably transfected with Pax8 splicing isoforms. Tg expression was analyzed in two different pools of clones for each transfected DNA. The GAPDH hybridization was used to normalize the amount of RNA present in each lane. ( B ) Reverse transcription–PCR analysis of cDNA derived from total RNA from the same cells as in A . Pax8 expression was analyzed in two different pools of clones for each transfected DNA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Pax8 has a key role in thyroid cell differentiation

    doi:

    Figure Lengend Snippet: Analysis of the transcriptional properties of Pax8 splicing isoforms. ( A ) Northern blot analysis of total RNA prepared from PCPy cells stably transfected with Pax8 splicing isoforms. Tg expression was analyzed in two different pools of clones for each transfected DNA. The GAPDH hybridization was used to normalize the amount of RNA present in each lane. ( B ) Reverse transcription–PCR analysis of cDNA derived from total RNA from the same cells as in A . Pax8 expression was analyzed in two different pools of clones for each transfected DNA.

    Article Snippet: To obtain stable clones, calcium phosphate–DNA precipitates were prepared with 10 μg of plasmid DNA containing either pCMV-Pax8 or ΔMT-Pax8 and 1 μg of a plasmid containing the neomycin resistance gene under the RSV promoter and 40 μg of calf thymus genomic DNA as a carrier (Boehringer Mannheim).

    Techniques: Northern Blot, Stable Transfection, Transfection, Expressing, Clone Assay, Hybridization, Polymerase Chain Reaction, Derivative Assay

    Fig. 2. hHR6A is a CDK substrate in vitro and is phosphorylated during the G 2 /M cell cycle phase in vivo . ( A ) Purified His 6 -hHR6A (top panel) or GST–pRb 773–928 (bottom panel) was incubated in the presence of [γ- 32 P]ATP, in either the absence (Control) or presence of purified cyclin D1–CDK4, cyclin E–CDK2, cyclin A–CDK2, cyclin A–CDK1 or cyclin B–CDK1, resolved by SDS–PAGE and visualized by autoradiography. ( B ) CHO cells transfected with either pCMV-Tag2 vector (Control) or pCMV-Tag2-hHR6A (hHR6A) were labelled with [ 32 P]phosphate and incubated with either dimethylsulfoxide (left and middle lanes) or 50 µM roscovitine (right lane) during the labelling period. Following labelling, hHR6A was immunoprecipitated, separated by SDS–PAGE and visualized by autoradiography. ( C ) CHO cells transfected with pCMV-Tag2-hHR6A (hHR6A) were synchronized by blocking in the G 2 /M cell cycle phase with nocodazole, then initiated to re-enter the cell cycle by nocodazole removal, harvested at various time points for DNA analysis by flow cytometry, and the proportion of cells in G 1 , S and G 2 M phases determined. ( D ) CHO cells transfected with either pCMV-Tag2 vector (Control) or pCMV-Tag2-hHR6A (hHR6A) were synchronized by blocking in the G 2 /M cell cycle phase with nocodazole (time = 0), initiated to re-enter the cell cycle by nocodazole removal and then lysed at various time points (as indicated). The cells were pulse-labelled with [ 32 P]phosphate for 3 h prior to lysis. Following lysis, hHR6A was immunoprecipitated, separated by SDS–PAGE and visualized by autoradiography (upper panel) or western blotting with an anti-FLAG antibody (lower panel). Phosphorylated hHR6A and the co-immunoprecipitating phosphoprotein (asterisk) are indicated with arrows.

    Journal: The EMBO Journal

    Article Title: Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation

    doi: 10.1093/emboj/21.8.2009

    Figure Lengend Snippet: Fig. 2. hHR6A is a CDK substrate in vitro and is phosphorylated during the G 2 /M cell cycle phase in vivo . ( A ) Purified His 6 -hHR6A (top panel) or GST–pRb 773–928 (bottom panel) was incubated in the presence of [γ- 32 P]ATP, in either the absence (Control) or presence of purified cyclin D1–CDK4, cyclin E–CDK2, cyclin A–CDK2, cyclin A–CDK1 or cyclin B–CDK1, resolved by SDS–PAGE and visualized by autoradiography. ( B ) CHO cells transfected with either pCMV-Tag2 vector (Control) or pCMV-Tag2-hHR6A (hHR6A) were labelled with [ 32 P]phosphate and incubated with either dimethylsulfoxide (left and middle lanes) or 50 µM roscovitine (right lane) during the labelling period. Following labelling, hHR6A was immunoprecipitated, separated by SDS–PAGE and visualized by autoradiography. ( C ) CHO cells transfected with pCMV-Tag2-hHR6A (hHR6A) were synchronized by blocking in the G 2 /M cell cycle phase with nocodazole, then initiated to re-enter the cell cycle by nocodazole removal, harvested at various time points for DNA analysis by flow cytometry, and the proportion of cells in G 1 , S and G 2 M phases determined. ( D ) CHO cells transfected with either pCMV-Tag2 vector (Control) or pCMV-Tag2-hHR6A (hHR6A) were synchronized by blocking in the G 2 /M cell cycle phase with nocodazole (time = 0), initiated to re-enter the cell cycle by nocodazole removal and then lysed at various time points (as indicated). The cells were pulse-labelled with [ 32 P]phosphate for 3 h prior to lysis. Following lysis, hHR6A was immunoprecipitated, separated by SDS–PAGE and visualized by autoradiography (upper panel) or western blotting with an anti-FLAG antibody (lower panel). Phosphorylated hHR6A and the co-immunoprecipitating phosphoprotein (asterisk) are indicated with arrows.

    Article Snippet: CHO cells were seeded at 1.0 × 106 cells/150 mm dish and transfected with 30 µg of plasmid DNA using FuGENE 6 (Boehringer Mannheim) according to the manufacturer’s instructions.

    Techniques: In Vitro, In Vivo, Purification, Incubation, SDS Page, Autoradiography, Transfection, Plasmid Preparation, Immunoprecipitation, Blocking Assay, Flow Cytometry, Cytometry, Lysis, Western Blot