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Boehringer Mannheim dna polymerase
Dna Polymerase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dna polymerase - by Bioz Stars, 2020-03
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Clone Assay:

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. To produce a promoter–luciferase construct with the entire cloned putative xC/EBPα promoter region, recombinant pUC18 containing the 1.5 kb Pst I fragment (see above) was used as a template for PCR with the M13 universal primer and an oligonucleotide designed against the +41 to +22 region of the xC/EBPα gene (designated as xalpha).

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The trailer was digested with Not I and Eco RI and, afterward, was cloned into the pGEM-leader construct. .. The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C).

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim). .. We used oligonucleotide primers for cDNA cloning and quantitative real-time RT-PCR analysis are listed in Tables and .

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: .. In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used. .. The reactions were stopped by the addition of formamide containing bromophenol blue and xylene cyanol dye, heated to 95" for 3 min, and chilled prior to electrophoresis in So/o polyacrylamide gels containing 7 M urea.

Centrifugation:

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: This mixture was incubated at room temperature for 5 min, precipitated with 3 vol of ethanol in the presence of 37.5 mM Na acetate pH5.5, and then collected by centrifugation in a microfuge for 5 min. .. In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used.

Amplification:

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: .. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim). .. The PCR fragments were subcloned into a TA vector (Invitrogen).

Article Title: Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics
Article Snippet: Amplification reactions were performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA, U.S.A.) with a ‘hot start' to help ensure amplification specificity. .. The 50 μ l reaction mixture contained 2.5 U of DNA polymerase, 5 μ l of a 10-fold diluted DNA sample (160–190 ng DNA), 12.5 pmol of each primer (7 pmol for exon 7), 0.05 m M dNTP (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and 5 μ l of 10 × PCR buffer containing 15 m M MgCl2 (Perkin-Elmer).

DNA Synthesis:

Article Title: Rolling Circle DNA Synthesis: Small Circular Oligonucleotides as Efficient Templates for DNA Polymerases
Article Snippet: .. Unless noted otherwise in the text, the conditions for DNA synthesis using the Klenow fragment (KF) of DNA Polymerase I were as follows: 10 nM circle, 10 nM primer, 1mM each of dATP, dTTP, dCTP, and dGTP (Boehringer Mannheim), and 4 units of KF (United States Biochemical) were incubated in 20 µL of Klenow reaction buffer (Tris•HCl 50 mM (pH 7.5), 10 mM MgCl2 , 1 mM dithiothreitol, 50 mg/mL BSA) for 1–24 h. The reaction was stopped by addition of 10 µL of a 40% formamide, 50 mM EDTA stop solution. .. For other enzymes, the conditions used were as follows: T4 DNA Polymerase (GIBCO BRL) 0.25 units/µL, 10 nM circle, 10 nM primer, 1 mM of each of the four dNTP’s in a buffer containing 33 mM Tris•acetate (pH 7.9), 66 mM sodium acetate, 10 mM magnesium acetate, 100 mg/mL BSA, 0.5 mM DTT, and with incubation at 37 °C for 3 h. T7 DNA Polymerase and Sequenase 2.0 (T7 Pol exonuclease free) obtained from United States Biochemical: 0.3 units/µL, 10 nM circle, 10 nM primer, 10 nM of each of the dNTP’s in a buffer containing 40 mM Tris•HCl (pH 7.5), 10 mM MgCl2 , 50 mM NaCl, 1 mM dithiothreitol, and with incubation at 37 °C for 3 h. Taq DNA Polymerase (GIBCO BRL) 0.25 units/µL, 10 nM circle, 10 nM primer, 1 mM of each of the dNTP’s in a buffer containing 20 mM Tris•HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2 , and with incubation at 65 °C for 3 h. E. Coli DNA Polymerase I (United States Biochemical) 0.5 units/µL, 10 nM circle, 10 nM primer, 1 mM of each of the dNTP’s in a buffer containing 20 mM Tris•HCl (pH 7.5), 10 mM MgCl2 , and with incubation at 37 °C for 3 h. Reactions were stopped as described above.

Construct:

Article Title: Central P2X4 and P2X6 Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor
Article Snippet: Wild-type full-length P2X6 subunit cDNA was obtained by RT-PCR using adult rat spinal cord RT-cDNA template, Expand DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and exact match primers based on published primary sequences ( ; ). .. To generate epitope-tagged P2X6 -Flag and P2X4 -(His)6 subunits, an Xho I– Xba I cassette containing an in-frame His6 epitope followed by an artificial stop codon was grafted to the full-length Hin dIII– Xho I P2X4 construct.

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: Paragraph title: Preparation of manipulated xC/EBPα promoter–reporter constructs ... The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ).

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The trailer was digested with Not I and Eco RI and, afterward, was cloned into the pGEM-leader construct. .. The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C).

Article Title: Environmental Regulation of Bacillus subtilis ?D-Dependent Gene Expression
Article Snippet: An in-frame translational fusion containing 180 bp of DNA upstream of the hag transcriptional start site and encoding a fusion protein comprising the first 71 amino acids of B. subtilis flagellin fused to the sixth amino acid (Gly) of the E. coli β-galactosidase enzyme was constructed and confirmed as follows. .. Cla I leaves a 5′ overhang ( Pst I does not), allowing for the fill-in reaction by the Klenow fragment of DNA polymerase (Boehringer Mannheim) and dCTP (Pharmacia) solely at this end.

Real-time Polymerase Chain Reaction:

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: Real time PCR primers were designed using sequence data. .. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim).

Incubation:

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: This mixture was incubated at room temperature for 5 min, precipitated with 3 vol of ethanol in the presence of 37.5 mM Na acetate pH5.5, and then collected by centrifugation in a microfuge for 5 min. .. In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used.

Article Title: Rolling Circle DNA Synthesis: Small Circular Oligonucleotides as Efficient Templates for DNA Polymerases
Article Snippet: .. Unless noted otherwise in the text, the conditions for DNA synthesis using the Klenow fragment (KF) of DNA Polymerase I were as follows: 10 nM circle, 10 nM primer, 1mM each of dATP, dTTP, dCTP, and dGTP (Boehringer Mannheim), and 4 units of KF (United States Biochemical) were incubated in 20 µL of Klenow reaction buffer (Tris•HCl 50 mM (pH 7.5), 10 mM MgCl2 , 1 mM dithiothreitol, 50 mg/mL BSA) for 1–24 h. The reaction was stopped by addition of 10 µL of a 40% formamide, 50 mM EDTA stop solution. .. For other enzymes, the conditions used were as follows: T4 DNA Polymerase (GIBCO BRL) 0.25 units/µL, 10 nM circle, 10 nM primer, 1 mM of each of the four dNTP’s in a buffer containing 33 mM Tris•acetate (pH 7.9), 66 mM sodium acetate, 10 mM magnesium acetate, 100 mg/mL BSA, 0.5 mM DTT, and with incubation at 37 °C for 3 h. T7 DNA Polymerase and Sequenase 2.0 (T7 Pol exonuclease free) obtained from United States Biochemical: 0.3 units/µL, 10 nM circle, 10 nM primer, 10 nM of each of the dNTP’s in a buffer containing 40 mM Tris•HCl (pH 7.5), 10 mM MgCl2 , 50 mM NaCl, 1 mM dithiothreitol, and with incubation at 37 °C for 3 h. Taq DNA Polymerase (GIBCO BRL) 0.25 units/µL, 10 nM circle, 10 nM primer, 1 mM of each of the dNTP’s in a buffer containing 20 mM Tris•HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2 , and with incubation at 65 °C for 3 h. E. Coli DNA Polymerase I (United States Biochemical) 0.5 units/µL, 10 nM circle, 10 nM primer, 1 mM of each of the dNTP’s in a buffer containing 20 mM Tris•HCl (pH 7.5), 10 mM MgCl2 , and with incubation at 37 °C for 3 h. Reactions were stopped as described above.

Expressing:

Article Title: Central P2X4 and P2X6 Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor
Article Snippet: Wild-type full-length P2X6 subunit cDNA was obtained by RT-PCR using adult rat spinal cord RT-cDNA template, Expand DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and exact match primers based on published primary sequences ( ; ). .. The P2X4 -(His)6 mutant was then subcloned directionally into the Hin dIII and Xba I sites of pcDNAI vector (Invitrogen, San Diego, CA) for cytomegalovirus-driven heterologous expression in mammalian cells and Xenopus laevis oocytes.

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: Real time RT-PCR The levels of MMP-2 and -9 gene expression were analyzed with real time RT-PCR using a Taqman probe as described previously [ ]. .. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim).

Transformation Assay:

Article Title: Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture
Article Snippet: Restriction enzymes, DNA polymerase, and T4 DNA ligase were obtained from and used as recommended by Boehringer Mannheim (Indianapolis, Ind.) and New England Biolabs (Beverly, Mass.). .. Plasmids were transformed into E. coli by electroporation using an E. coli Pulser (Bio-Rad Inc., Hercules, Calif.) with a field strength of 18 kV/cm.

Chloramphenicol Acetyltransferase Assay:

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C). .. As a reporter gene, 668 nt of the CAT gene, flanked by Not I sites, was inserted between the leader and trailer sequences.

Article Title: Comparison of Genetic Divergence and Fitness between Two Subclones of Helicobacter pylori
Article Snippet: .. The reaction mixture contained PCR buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 100 μM concentrations of deoxynucleoside triphosphates, 0.5 U of DNA polymerase (Boehringer Mannheim), 7 μM concentrations of primers UpCagF (ACT TTC ACG CCC TTT CCC TCC) and DownCagR (TTG CAT GCG TTA TTA TTT CAC), and H. pylori genomic DNA. ..

Hybridization:

Article Title: The Two Forms of Karyogamy Transcription Factor Kar4p Are Regulated by Differential Initiation of Transcription, Translation, and Protein Turnover
Article Snippet: After extension with the Klenow fragment from DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.), the product was cleaved with Sac I to delineate the 3′ end of the probe. .. After hybridization of the probe with the yeast RNA, 400 U of S1 nuclease (Boehringer Mannheim) was used.

Countercurrent Chromatography:

Article Title: Comparison of Genetic Divergence and Fitness between Two Subclones of Helicobacter pylori
Article Snippet: .. The reaction mixture contained PCR buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 100 μM concentrations of deoxynucleoside triphosphates, 0.5 U of DNA polymerase (Boehringer Mannheim), 7 μM concentrations of primers UpCagF (ACT TTC ACG CCC TTT CCC TCC) and DownCagR (TTG CAT GCG TTA TTA TTT CAC), and H. pylori genomic DNA. ..

Electroporation:

Article Title: Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture
Article Snippet: Restriction enzymes, DNA polymerase, and T4 DNA ligase were obtained from and used as recommended by Boehringer Mannheim (Indianapolis, Ind.) and New England Biolabs (Beverly, Mass.). .. Plasmids were transformed into E. coli by electroporation using an E. coli Pulser (Bio-Rad Inc., Hercules, Calif.) with a field strength of 18 kV/cm.

Oligonucleotide Synthesis:

Article Title: Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture
Article Snippet: Restriction enzymes, DNA polymerase, and T4 DNA ligase were obtained from and used as recommended by Boehringer Mannheim (Indianapolis, Ind.) and New England Biolabs (Beverly, Mass.). .. Oligonucleotide synthesis and sequencing were done by Genemed (South San Francisco, Calif.).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Central P2X4 and P2X6 Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor
Article Snippet: .. Wild-type full-length P2X6 subunit cDNA was obtained by RT-PCR using adult rat spinal cord RT-cDNA template, Expand DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and exact match primers based on published primary sequences ( ; ). ..

Generated:

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: A 439-nt fragment of the 5′ end (trailer) was generated the same way under the same conditions. .. The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C).

other:

Article Title: The Cell Cycle-Specific Growth-Inhibitory Factor Produced by Actinobacillus actinomycetemcomitans Is a Cytolethal Distending Toxin
Article Snippet: All restriction enzymes, T4 DNA ligase, and Klenow fragment of DNA polymerase I were from Boehringer Mannheim, Tokyo, Japan, or New England BioLabs, Inc., Beverly, Mass.

DNA Sequencing:

Article Title: The Two Forms of Karyogamy Transcription Factor Kar4p Are Regulated by Differential Initiation of Transcription, Translation, and Protein Turnover
Article Snippet: After extension with the Klenow fragment from DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.), the product was cleaved with Sac I to delineate the 3′ end of the probe. .. Sequencing reactions were carried out according to the Sequenase, version 2.0, protocol (Sequenase DNA sequencing kit; United States Biochemicals).

Article Title: Amide-Based Prodrugs of Spermidine-Bridged Dinuclear Platinum. Synthesis, DNA Binding, and Biological Activity
Article Snippet: EcoR I and Nde I restriction endonucleases, thermal cycle dideoxy DNA sequencing kit with VentR (exo+ ) DNA polymerases, and T4 polynucleotide kinase were purchased from New England Biolabs. .. Klenow fragment of DNA polymerase I was from Boehringer-Mannheim Biochemica.

Article Title: Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics
Article Snippet: M13 ‘tags' were added to the 5′-ends of each primer to make it possible to use dye-primer DNA-sequencing chemistry to facilitate the identification of heterozygous bases ( ). .. The 50 μ l reaction mixture contained 2.5 U of DNA polymerase, 5 μ l of a 10-fold diluted DNA sample (160–190 ng DNA), 12.5 pmol of each primer (7 pmol for exon 7), 0.05 m M dNTP (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and 5 μ l of 10 × PCR buffer containing 15 m M MgCl2 (Perkin-Elmer).

Polymerase Chain Reaction:

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: .. The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. To produce a promoter–luciferase construct with the entire cloned putative xC/EBPα promoter region, recombinant pUC18 containing the 1.5 kb Pst I fragment (see above) was used as a template for PCR with the M13 universal primer and an oligonucleotide designed against the +41 to +22 region of the xC/EBPα gene (designated as xalpha).

Article Title: Human 3?-Hydroxysteroid Dehydrogenase Types 1 and 2: Gene Sequence Variation and Functional Genomics
Article Snippet: .. All amplifications were performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA), and the 25 µl reaction mixtures contained 0.75 U of DNA polymerase, 0.5 µl of a 10-fold diluted DNA sample (16–19 ng DNA per reaction), 5 to 10 pmol of each primer, 0.08 mM dNTP (Boehringer Mannheim, Indianapolis, IN), 0 or 2% DMSO and 2.5 µl of 10X PCR buffer containing 15 mM MgCl2 (Perkin-Elmer). .. Amplicons were sequenced on both strands in the Mayo Molecular Biology Core Facility with an ABI 377 DNA sequencer using BigDye (Perkin-Elmer) dye primer sequencing chemistry.

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The primer for cDNA synthesis, flanked by a Not I site, was complementary to nt 18670 to 18698 of the vRNA; the second primer for PCR was homologous to the last 32 nt of the 5′ end and contained Nco I and Eco RI restriction sites. .. The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C).

Article Title: Comparison of Genetic Divergence and Fitness between Two Subclones of Helicobacter pylori
Article Snippet: .. The reaction mixture contained PCR buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 100 μM concentrations of deoxynucleoside triphosphates, 0.5 U of DNA polymerase (Boehringer Mannheim), 7 μM concentrations of primers UpCagF (ACT TTC ACG CCC TTT CCC TCC) and DownCagR (TTG CAT GCG TTA TTA TTT CAC), and H. pylori genomic DNA. ..

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: .. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim). .. The PCR fragments were subcloned into a TA vector (Invitrogen).

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: Paragraph title: Sequencing of cloned DNA and PCR fragments ... In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used.

Article Title: Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics
Article Snippet: .. The 50 μ l reaction mixture contained 2.5 U of DNA polymerase, 5 μ l of a 10-fold diluted DNA sample (160–190 ng DNA), 12.5 pmol of each primer (7 pmol for exon 7), 0.05 m M dNTP (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and 5 μ l of 10 × PCR buffer containing 15 m M MgCl2 (Perkin-Elmer). .. PCR cycling parameters involved a 12 min ‘hot start' at 94°C, followed by 35 cycles (40 cycles for exon 7) of 94°C for 30 s, 55°C (68°C for the exon 7) for 30 s and 45 s at 72°C– with a final 10 min extension at 72°C.

Recombinant:

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. To produce a promoter–luciferase construct with the entire cloned putative xC/EBPα promoter region, recombinant pUC18 containing the 1.5 kb Pst I fragment (see above) was used as a template for PCR with the M13 universal primer and an oligonucleotide designed against the +41 to +22 region of the xC/EBPα gene (designated as xalpha).

Mutagenesis:

Article Title: Central P2X4 and P2X6 Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor
Article Snippet: Wild-type full-length P2X6 subunit cDNA was obtained by RT-PCR using adult rat spinal cord RT-cDNA template, Expand DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and exact match primers based on published primary sequences ( ; ). .. The P2X4 -(His)6 mutant was then subcloned directionally into the Hin dIII and Xba I sites of pcDNAI vector (Invitrogen, San Diego, CA) for cytomegalovirus-driven heterologous expression in mammalian cells and Xenopus laevis oocytes.

Article Title: Human 3?-Hydroxysteroid Dehydrogenase Types 1 and 2: Gene Sequence Variation and Functional Genomics
Article Snippet: All amplifications were performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA), and the 25 µl reaction mixtures contained 0.75 U of DNA polymerase, 0.5 µl of a 10-fold diluted DNA sample (16–19 ng DNA per reaction), 5 to 10 pmol of each primer, 0.08 mM dNTP (Boehringer Mannheim, Indianapolis, IN), 0 or 2% DMSO and 2.5 µl of 10X PCR buffer containing 15 mM MgCl2 (Perkin-Elmer). .. Chromatograms were analyzed using the University of Washington PolyPhred 3.0 and Consed 8.0 programs [ , ] as well as with Mutation Surveyor (SoftGenetics, LLC, State College, PA).

Isolation:

Article Title: Amide-Based Prodrugs of Spermidine-Bridged Dinuclear Platinum. Synthesis, DNA Binding, and Biological Activity
Article Snippet: Plasmids pSP73 (2464 base pairs) were isolated according to standard procedures. .. Klenow fragment of DNA polymerase I was from Boehringer-Mannheim Biochemica.

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: Partial bovine MMP-2 and -9 mRNA strands were amplified and identified as follows: Total RNA was isolated using ISOGEN (Nippon Gene, Kyoto, Japan), and one microgram of total RNA was subjected to reverse transcription of cDNA with transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. .. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim).

Article Title: Environmental Regulation of Bacillus subtilis ?D-Dependent Gene Expression
Article Snippet: Cla I leaves a 5′ overhang ( Pst I does not), allowing for the fill-in reaction by the Klenow fragment of DNA polymerase (Boehringer Mannheim) and dCTP (Pharmacia) solely at this end. .. After these manipulations, the 5.1-kb pDM67 fragment containing the transcriptional and translational start site information for hag in pJM102 ( ) was isolated from the above-described 2.38-kb fragment by gel purification (Geneclean kit; BIO 101, Inc.).

Subcloning:

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: .. The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. To produce a promoter–luciferase construct with the entire cloned putative xC/EBPα promoter region, recombinant pUC18 containing the 1.5 kb Pst I fragment (see above) was used as a template for PCR with the M13 universal primer and an oligonucleotide designed against the +41 to +22 region of the xC/EBPα gene (designated as xalpha).

Labeling:

Article Title: The Two Forms of Karyogamy Transcription Factor Kar4p Are Regulated by Differential Initiation of Transcription, Translation, and Protein Turnover
Article Snippet: The primer was end labeled and hybridized to the KAR4 DNA fragment. .. After extension with the Klenow fragment from DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.), the product was cleaved with Sac I to delineate the 3′ end of the probe.

Purification:

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: In a typical experiment, 4 pg of circular plasmid DNA or purified PCR fragment DNA was added to 40 ~1 of 0.2 M NaOH, 0.2 mM EDTA in order to separate the strands. .. In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used.

Article Title: Environmental Regulation of Bacillus subtilis ?D-Dependent Gene Expression
Article Snippet: Cla I leaves a 5′ overhang ( Pst I does not), allowing for the fill-in reaction by the Klenow fragment of DNA polymerase (Boehringer Mannheim) and dCTP (Pharmacia) solely at this end. .. This 5.1-kb blunt-end Pst I fragment was then ligated to the similarly purified 3.1-kb Sma I- Pst I fragment of pMC1871 bearing lacZ .

Sequencing:

Article Title: The Two Forms of Karyogamy Transcription Factor Kar4p Are Regulated by Differential Initiation of Transcription, Translation, and Protein Turnover
Article Snippet: Reaction products were ethanol precipitated, resuspended in sample buffer, and then run on a denaturing polyacrylamide-urea gel in parallel with sequencing reactions (see below). .. After extension with the Klenow fragment from DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.), the product was cleaved with Sac I to delineate the 3′ end of the probe.

Article Title: Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture
Article Snippet: Restriction enzymes, DNA polymerase, and T4 DNA ligase were obtained from and used as recommended by Boehringer Mannheim (Indianapolis, Ind.) and New England Biolabs (Beverly, Mass.). .. Oligonucleotide synthesis and sequencing were done by Genemed (South San Francisco, Calif.).

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: .. The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. To produce a promoter–luciferase construct with the entire cloned putative xC/EBPα promoter region, recombinant pUC18 containing the 1.5 kb Pst I fragment (see above) was used as a template for PCR with the M13 universal primer and an oligonucleotide designed against the +41 to +22 region of the xC/EBPα gene (designated as xalpha).

Article Title: Human 3?-Hydroxysteroid Dehydrogenase Types 1 and 2: Gene Sequence Variation and Functional Genomics
Article Snippet: Specifically, a long PCR was performed, followed by two nested amplifications to obtain 5′-FR sequence as well as the sequences of exons 1 and 2. .. All amplifications were performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA), and the 25 µl reaction mixtures contained 0.75 U of DNA polymerase, 0.5 µl of a 10-fold diluted DNA sample (16–19 ng DNA per reaction), 5 to 10 pmol of each primer, 0.08 mM dNTP (Boehringer Mannheim, Indianapolis, IN), 0 or 2% DMSO and 2.5 µl of 10X PCR buffer containing 15 mM MgCl2 (Perkin-Elmer).

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C). .. For generating negative-strand RNA, the 5′ end of the MBGV minigenome was positioned adjacent to the T7 RNA polymerase promoter and the 3′ end was followed by the hepatitis delta virus ribozyme sequence (Fig. A).

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: Real time PCR primers were designed using sequence data. .. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim).

Article Title: Characterization of Sparsomycin Resistance in Streptomyces sparsogenes
Article Snippet: T4 DNA ligase, calf intestinal alkaline phosphatase, and the DNA polymerase I Klenow fragment were from Boehringer Mannheim. .. Nucleotide sequences of DNA inserts were determined on both strands using a Dye-Terminator cycle sequencing ready reaction kit (Applied Biosystems) with custom-made oligonucleotides as primers.

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: .. In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used. .. The reactions were stopped by the addition of formamide containing bromophenol blue and xylene cyanol dye, heated to 95" for 3 min, and chilled prior to electrophoresis in So/o polyacrylamide gels containing 7 M urea.

Article Title: Human estrogen sulfotransferase (SULT1E1) pharmacogenomics: gene resequencing and functional genomics
Article Snippet: Primers were designed to hybridize within introns, within the 5′-flanking region or within the 3′-untranslated region of the terminal exon at locations selected to avoid repetitive sequence. .. The 50 μ l reaction mixture contained 2.5 U of DNA polymerase, 5 μ l of a 10-fold diluted DNA sample (160–190 ng DNA), 12.5 pmol of each primer (7 pmol for exon 7), 0.05 m M dNTP (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and 5 μ l of 10 × PCR buffer containing 15 m M MgCl2 (Perkin-Elmer).

Quantitative RT-PCR:

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: Paragraph title: Real time RT-PCR ... After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim).

Activated Clotting Time Assay:

Article Title: Comparison of Genetic Divergence and Fitness between Two Subclones of Helicobacter pylori
Article Snippet: .. The reaction mixture contained PCR buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 100 μM concentrations of deoxynucleoside triphosphates, 0.5 U of DNA polymerase (Boehringer Mannheim), 7 μM concentrations of primers UpCagF (ACT TTC ACG CCC TTT CCC TCC) and DownCagR (TTG CAT GCG TTA TTA TTT CAC), and H. pylori genomic DNA. ..

Plasmid Preparation:

Article Title: Central P2X4 and P2X6 Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor
Article Snippet: Wild-type full-length P2X6 subunit cDNA was obtained by RT-PCR using adult rat spinal cord RT-cDNA template, Expand DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and exact match primers based on published primary sequences ( ; ). .. The P2X4 -(His)6 mutant was then subcloned directionally into the Hin dIII and Xba I sites of pcDNAI vector (Invitrogen, San Diego, CA) for cytomegalovirus-driven heterologous expression in mammalian cells and Xenopus laevis oocytes.

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: .. The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. To produce a promoter–luciferase construct with the entire cloned putative xC/EBPα promoter region, recombinant pUC18 containing the 1.5 kb Pst I fragment (see above) was used as a template for PCR with the M13 universal primer and an oligonucleotide designed against the +41 to +22 region of the xC/EBPα gene (designated as xalpha).

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The leader was digested with Sph I and Bam HI and cloned into the vector pGEM3Zf(+). .. The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C).

Article Title: Gelatinase (MMP-2 and -9) expression profiles during gestation in the bovine endometrium
Article Snippet: After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim). .. The PCR fragments were subcloned into a TA vector (Invitrogen).

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: In a typical experiment, 4 pg of circular plasmid DNA or purified PCR fragment DNA was added to 40 ~1 of 0.2 M NaOH, 0.2 mM EDTA in order to separate the strands. .. In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used.

Article Title: Environmental Regulation of Bacillus subtilis ?D-Dependent Gene Expression
Article Snippet: The 2.38-kb Cla I- Pst I fragment of plasmid pDM67 , containing most of the coding region for hag , was replaced with the 3.1-kb Sma I- Pst I fragment of pMC1871 that contains the lacZ coding region ( ). .. Cla I leaves a 5′ overhang ( Pst I does not), allowing for the fill-in reaction by the Klenow fragment of DNA polymerase (Boehringer Mannheim) and dCTP (Pharmacia) solely at this end.

Electrophoresis:

Article Title: Comparison of a dengue-2 virus and its candidate vaccine derivative: sequence relationships with the flaviviruses and other viruses.
Article Snippet: In most cases 0.5 unit DNA polymerase (Klenow fragment, Boehringer-Mannheim) was used to obtain sequence data, but for some of the larger clones, 3 units of Sequenase (Trace) was used. .. The reactions were stopped by the addition of formamide containing bromophenol blue and xylene cyanol dye, heated to 95" for 3 min, and chilled prior to electrophoresis in So/o polyacrylamide gels containing 7 M urea.

Agarose Gel Electrophoresis:

Article Title: Characterization of Sparsomycin Resistance in Streptomyces sparsogenes
Article Snippet: Restriction enzyme digestions, ligations, agarose gel electrophoresis, etc., were performed according to well-established techniques ( ). .. T4 DNA ligase, calf intestinal alkaline phosphatase, and the DNA polymerase I Klenow fragment were from Boehringer Mannheim.

Produced:

Article Title: Analysis of the Xenopus laevis CCAAT-enhancer binding protein ? gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1
Article Snippet: The PCR reactions were performed using either Vent DNA polymerase or the ExpandTM system (Boehringer Mannheim), in order to minimise PCR-generated mutations, and this was confirmed by sequencing of all the products following subcloning into the pGEM-T vector ( ). .. Three further deletions were produced similarly using a common xalpha primer and oligonucleotides designed against the –634 to -615, –321 to –302 and –121 to –108 regions, respectively.

Concentration Assay:

Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes
Article Snippet: The leader-trailer DNA was excised with Nco I, and the overlapping 5′ ends (5′-CATG) were partially filled with dCTP, using the Klenow fragment of DNA polymerase (Boehringer Mannheim; 20 min at 22°C). .. Thereafter, the remaining overlapping 5′ ends were removed by nuclease S1 (Boehringer Mannheim) treatment (final concentration, 66 mU/μl; 5 min at 37°C), and the fragment was inserted between the Stu I and Sma I sites of the transcription vector 2,0 (kindly provided by Andrew Ball, University of Alabama Medical School) ( ).

Gel Purification:

Article Title: Environmental Regulation of Bacillus subtilis ?D-Dependent Gene Expression
Article Snippet: Cla I leaves a 5′ overhang ( Pst I does not), allowing for the fill-in reaction by the Klenow fragment of DNA polymerase (Boehringer Mannheim) and dCTP (Pharmacia) solely at this end. .. After these manipulations, the 5.1-kb pDM67 fragment containing the transcriptional and translational start site information for hag in pJM102 ( ) was isolated from the above-described 2.38-kb fragment by gel purification (Geneclean kit; BIO 101, Inc.).

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  • 83
    Boehringer Mannheim tth dna polymerase
    The analysis of <t>DNA</t> product synthesized by <t>Tth</t> and Δ Tth DNA polymerases by restriction enzymes. The DNA product synthesized in the standard reaction mixture at 74°C for 30 min by Tth or Δ Tth DNA polymerase in the presence of 1 µg/ml of the indicated oligoDNA (third line) was electrophoresed on an agarose gel under non-denaturing conditions before (lanes 1, 3, 5 and 7) or after (lanes 2, 4, 6 and 8) treatment with 1 U/ml of the restriction enzyme indicated at the bottom. The size markers are shown in kb on the left. The recognition sequence of the restriction enzyme is shown below each enzyme.
    Tth Dna Polymerase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 83/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tth dna polymerase/product/Boehringer Mannheim
    Average 83 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    tth dna polymerase - by Bioz Stars, 2020-03
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    89
    Boehringer Mannheim high fidelity dna polymerase
    The cDNA expression system with IRES-derived expression plasmids. (A) Constructs of IRES-derived plasmids used for the expression of two subunits simultaneously. (B) Time course of <t>DNA</t> polymerase activity in the cell extracts transfected with various constructs. COS-1 cells were transfected with pcDEBΔ (■), pSRα180 alone (⧫), pSRα180 and pSRα68 (●), or pI-pol.α (▴); incubated for 24, 48, and 72 h; and then lysed with a solution containing 20 mM potassium phosphate (pH 7.5), 300 mM KCl, 10% glycerol, 0.05% Triton X-100, and 0.1 mM EDTA. After centrifugation, the supernatant was assayed to estimate DNA polymerase activity. Five micrograms of protein of COS-1 extracts was incubated with [ 3 H]dTTP and DNase I-activated calf thymus DNA for 1 h at 37°C, and incorporation of radioactive material was measured. (C) Subcellular distribution of transiently overexpressed subunits of DNA polymerase α-primase complex. pSRα68 (a), pSRα180 (b), pSRα180 and pSRα68 (c), pI-pol.α (d), pSRα54 (e), pSRα46-HA (f), pSRα54 and pSRα46-HA (g), or pI-pri(HA) (h) was transfected into COS-1 cells, and the proteins expressed were detected by immunofluorescence analysis with anti-p68 (a, c, and d) or anti-p54 (e, g, and h) polyclonal antibodies and FITC-conjugated anti-rabbit IgG antibody or SJK132-20 anti-p180 monoclonal antibody (b, c, and d) or <t>12CA5</t> anti-HA tag antibody (f, g, and h) and Texas red-conjugated anti-mouse IgG antibody. DNA was stained blue by Hoechst 33258, and pictures were merged.
    High Fidelity Dna Polymerase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high fidelity dna polymerase/product/Boehringer Mannheim
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    The analysis of DNA product synthesized by Tth and Δ Tth DNA polymerases by restriction enzymes. The DNA product synthesized in the standard reaction mixture at 74°C for 30 min by Tth or Δ Tth DNA polymerase in the presence of 1 µg/ml of the indicated oligoDNA (third line) was electrophoresed on an agarose gel under non-denaturing conditions before (lanes 1, 3, 5 and 7) or after (lanes 2, 4, 6 and 8) treatment with 1 U/ml of the restriction enzyme indicated at the bottom. The size markers are shown in kb on the left. The recognition sequence of the restriction enzyme is shown below each enzyme.

    Journal: Nucleic Acids Research

    Article Title: Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus

    doi:

    Figure Lengend Snippet: The analysis of DNA product synthesized by Tth and Δ Tth DNA polymerases by restriction enzymes. The DNA product synthesized in the standard reaction mixture at 74°C for 30 min by Tth or Δ Tth DNA polymerase in the presence of 1 µg/ml of the indicated oligoDNA (third line) was electrophoresed on an agarose gel under non-denaturing conditions before (lanes 1, 3, 5 and 7) or after (lanes 2, 4, 6 and 8) treatment with 1 U/ml of the restriction enzyme indicated at the bottom. The size markers are shown in kb on the left. The recognition sequence of the restriction enzyme is shown below each enzyme.

    Article Snippet: The amount of DNA synthesized was 12 pmol by Tth DNA polymerase and < 1 pmol by Δ Tth DNA polymerase, a mutant Tth DNA polymerase lacking 5′→3′ exonuclease activity , when no oligoDNA was added to the reaction mixture (Table ).

    Techniques: Synthesized, Agarose Gel Electrophoresis, Sequencing

    The cDNA expression system with IRES-derived expression plasmids. (A) Constructs of IRES-derived plasmids used for the expression of two subunits simultaneously. (B) Time course of DNA polymerase activity in the cell extracts transfected with various constructs. COS-1 cells were transfected with pcDEBΔ (■), pSRα180 alone (⧫), pSRα180 and pSRα68 (●), or pI-pol.α (▴); incubated for 24, 48, and 72 h; and then lysed with a solution containing 20 mM potassium phosphate (pH 7.5), 300 mM KCl, 10% glycerol, 0.05% Triton X-100, and 0.1 mM EDTA. After centrifugation, the supernatant was assayed to estimate DNA polymerase activity. Five micrograms of protein of COS-1 extracts was incubated with [ 3 H]dTTP and DNase I-activated calf thymus DNA for 1 h at 37°C, and incorporation of radioactive material was measured. (C) Subcellular distribution of transiently overexpressed subunits of DNA polymerase α-primase complex. pSRα68 (a), pSRα180 (b), pSRα180 and pSRα68 (c), pI-pol.α (d), pSRα54 (e), pSRα46-HA (f), pSRα54 and pSRα46-HA (g), or pI-pri(HA) (h) was transfected into COS-1 cells, and the proteins expressed were detected by immunofluorescence analysis with anti-p68 (a, c, and d) or anti-p54 (e, g, and h) polyclonal antibodies and FITC-conjugated anti-rabbit IgG antibody or SJK132-20 anti-p180 monoclonal antibody (b, c, and d) or 12CA5 anti-HA tag antibody (f, g, and h) and Texas red-conjugated anti-mouse IgG antibody. DNA was stained blue by Hoechst 33258, and pictures were merged.

    Journal: Molecular and Cellular Biology

    Article Title: Molecular Architecture of the Mouse DNA Polymerase ?-Primase Complex

    doi:

    Figure Lengend Snippet: The cDNA expression system with IRES-derived expression plasmids. (A) Constructs of IRES-derived plasmids used for the expression of two subunits simultaneously. (B) Time course of DNA polymerase activity in the cell extracts transfected with various constructs. COS-1 cells were transfected with pcDEBΔ (■), pSRα180 alone (⧫), pSRα180 and pSRα68 (●), or pI-pol.α (▴); incubated for 24, 48, and 72 h; and then lysed with a solution containing 20 mM potassium phosphate (pH 7.5), 300 mM KCl, 10% glycerol, 0.05% Triton X-100, and 0.1 mM EDTA. After centrifugation, the supernatant was assayed to estimate DNA polymerase activity. Five micrograms of protein of COS-1 extracts was incubated with [ 3 H]dTTP and DNase I-activated calf thymus DNA for 1 h at 37°C, and incorporation of radioactive material was measured. (C) Subcellular distribution of transiently overexpressed subunits of DNA polymerase α-primase complex. pSRα68 (a), pSRα180 (b), pSRα180 and pSRα68 (c), pI-pol.α (d), pSRα54 (e), pSRα46-HA (f), pSRα54 and pSRα46-HA (g), or pI-pri(HA) (h) was transfected into COS-1 cells, and the proteins expressed were detected by immunofluorescence analysis with anti-p68 (a, c, and d) or anti-p54 (e, g, and h) polyclonal antibodies and FITC-conjugated anti-rabbit IgG antibody or SJK132-20 anti-p180 monoclonal antibody (b, c, and d) or 12CA5 anti-HA tag antibody (f, g, and h) and Texas red-conjugated anti-mouse IgG antibody. DNA was stained blue by Hoechst 33258, and pictures were merged.

    Article Snippet: All restriction enzymes and Klenow fragment were purchased from Takara (Ohtsu, Japan); phenylmethylsulfonyl fluoride was from Sigma; Expand high-fidelity DNA polymerase and 12CA5 anti-hemagglutinin (HA) antibody were from Boehringer Mannheim; anti-T7 tag antibody was from Novagen; horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G (IgG) antibodies were from MBL (Nagoya, Japan); fluorescein isothiocyanate (FITC)- or Texas red-conjugated goat anti-rabbit or anti-mouse IgG antibodies were from Vector Inc.; fetal bovine serum was from Intergen; calf serum was from HyClone; anti-six-His monoclonal antibody and cobalt-chelating Sepharose (TALON) were from Clontech.

    Techniques: Expressing, Derivative Assay, Construct, Activity Assay, Transfection, Incubation, Centrifugation, Immunofluorescence, Staining