high fidelity polymerase chain reaction pcr system polymerase Search Results


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  • 99
    New England Biolabs high fidelity polymerase chain reaction pcr
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    Roche high fidelity pcr kit
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
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    Roche high fidelity polymerase chain reaction pcr system polymerase
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    High Fidelity Polymerase Chain Reaction Pcr System Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transformation and virulence of restriction mutants of S. aureus USA300 strain NRS384. (A) <t>PCR</t> profiles (primers IM110/IM111) of the sauUSI region amplified from NRS384 (lanes 1), NRS384 hsdR INT sauUSI INT (lanes 2), and NRS384 hsdR INT sauUSI ECORV (lanes 3) without or with EcoRV digestion. (B) Concentrated pRMC2 <t>DNA</t> (5 µg) isolated from E . coli DH10B ( dam + dcm + ) (light grey bars) or DC10B ( dam + ) (dark grey bars) was electroporated into the strains described above, and transformants were enumerated. (C) Intravenous injection of 2 × 10 6 CFU into 6- to 7-week-old female A/J mice. On day 7 of infection, the mice were euthanized, both kidneys were aseptically removed, and the bacterial CFU were enumerated as described in Materials and Methods. Each symbol represents the value for an individual mouse, and the short black line represents the mean for the group of mice. The broken line denotes the limit of detection at 333 CFU for the two kidneys.
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    Transformation and virulence of restriction mutants of S. aureus USA300 strain NRS384. (A) <t>PCR</t> profiles (primers IM110/IM111) of the sauUSI region amplified from NRS384 (lanes 1), NRS384 hsdR INT sauUSI INT (lanes 2), and NRS384 hsdR INT sauUSI ECORV (lanes 3) without or with EcoRV digestion. (B) Concentrated pRMC2 <t>DNA</t> (5 µg) isolated from E . coli DH10B ( dam + dcm + ) (light grey bars) or DC10B ( dam + ) (dark grey bars) was electroporated into the strains described above, and transformants were enumerated. (C) Intravenous injection of 2 × 10 6 CFU into 6- to 7-week-old female A/J mice. On day 7 of infection, the mice were euthanized, both kidneys were aseptically removed, and the bacterial CFU were enumerated as described in Materials and Methods. Each symbol represents the value for an individual mouse, and the short black line represents the mean for the group of mice. The broken line denotes the limit of detection at 333 CFU for the two kidneys.
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    <t>Adcy6</t> recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative <t>PCR</t> ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination
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    Image Search Results


    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Journal: Frontiers in Immunology

    Article Title: A Novel Pax5-Binding Regulatory Element in the Ig? Locus

    doi: 10.3389/fimmu.2014.00240

    Figure Lengend Snippet: Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Article Snippet: WT and ΔDm rearranged Igκ alleles were amplified with Vκ-Degenerate 5′-GTCCCTGCCAGGTTYAGTGGCAGTGGRTCWRGGAC-3′ and R3-1 5′-CAGACCCTGGTCTAATGGTTTGTAACCACATGGG-3′ primers using high fidelity PCR kit (Roche) with an initial denaturation of 4 min at 94°C, followed by 35 cycles of denaturation at 94°C for 15 s and annealing combined with elongation at 68°C for 2 min. 3′ A-overhang nucleotides were added by 20 min incubation with Taq polymerase and ATP at 72°C.

    Techniques: Binding Assay, Sequencing, Electro Mobility Shift Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Footprinting, Labeling, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction

    Transformation and virulence of restriction mutants of S. aureus USA300 strain NRS384. (A) PCR profiles (primers IM110/IM111) of the sauUSI region amplified from NRS384 (lanes 1), NRS384 hsdR INT sauUSI INT (lanes 2), and NRS384 hsdR INT sauUSI ECORV (lanes 3) without or with EcoRV digestion. (B) Concentrated pRMC2 DNA (5 µg) isolated from E . coli DH10B ( dam + dcm + ) (light grey bars) or DC10B ( dam + ) (dark grey bars) was electroporated into the strains described above, and transformants were enumerated. (C) Intravenous injection of 2 × 10 6 CFU into 6- to 7-week-old female A/J mice. On day 7 of infection, the mice were euthanized, both kidneys were aseptically removed, and the bacterial CFU were enumerated as described in Materials and Methods. Each symbol represents the value for an individual mouse, and the short black line represents the mean for the group of mice. The broken line denotes the limit of detection at 333 CFU for the two kidneys.

    Journal: mBio

    Article Title: Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

    doi: 10.1128/mBio.00277-11

    Figure Lengend Snippet: Transformation and virulence of restriction mutants of S. aureus USA300 strain NRS384. (A) PCR profiles (primers IM110/IM111) of the sauUSI region amplified from NRS384 (lanes 1), NRS384 hsdR INT sauUSI INT (lanes 2), and NRS384 hsdR INT sauUSI ECORV (lanes 3) without or with EcoRV digestion. (B) Concentrated pRMC2 DNA (5 µg) isolated from E . coli DH10B ( dam + dcm + ) (light grey bars) or DC10B ( dam + ) (dark grey bars) was electroporated into the strains described above, and transformants were enumerated. (C) Intravenous injection of 2 × 10 6 CFU into 6- to 7-week-old female A/J mice. On day 7 of infection, the mice were euthanized, both kidneys were aseptically removed, and the bacterial CFU were enumerated as described in Materials and Methods. Each symbol represents the value for an individual mouse, and the short black line represents the mean for the group of mice. The broken line denotes the limit of detection at 333 CFU for the two kidneys.

    Article Snippet: High-fidelity PCR was performed with KOD Hotstart DNA polymerase (Novagen) or Phusion DNA polymerase (Finnzymes) on genomic DNA isolated with the Genelute bacterial genomic DNA kit (Sigma).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Amplification, Isolation, Injection, Mouse Assay, Infection

    Schematic of allelic exchange with pIMAY. ( A ) A plasmid isolated from E . coli DC10B is transformed into staphylococci at 28°C, and single-crossover (SCO) integration was stimulated by growth at 37°C in the presence of chloramphenicol. The loss of replicating plasmid is assayed by colony PCR with MCS primers (IM151/IM152). Clones negative for replicating plasmid are then screened for the side of integration with a combination of chromosomal and cloning primers (e.g., OUT FWD/D REV [AB integration {AB INT}] or OUT REV/A FWD [CD integration {CD INT}]). The diagram details an integration event through the AB side (equivalent to clone h in panels B and C). A clone from either AB or CD integration event is grown at 28°C in broth without antibiotic selection to stimulate rolling circle replication and then plated on TSA with 1 µg/ml ATc. Expression of the secY antisense RNA ( a - secY ) inhibits growth of cells maintaining the plasmid. Plasmid excision through the AB side recreates the wild-type locus, while CD excision yields a mutated gene. (B) Colony PCR from 10 randomly chosen clones (clones a to j) after growth at 37°C for the presence of replicating plasmid. The absence of product indicates that the plasmid has integrated. Colony PCR from cells grown at 28°C is included as a positive control (+ve). (C) Two clones without replicating plasmid (clones c and h) were shown by colony PCR to have integrated either on the AB (upstream [clone h]) or CD (downstream [clone c]) side of the gene to be deleted. Wild-type (WT) genomic DNA was included as a control.

    Journal: mBio

    Article Title: Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

    doi: 10.1128/mBio.00277-11

    Figure Lengend Snippet: Schematic of allelic exchange with pIMAY. ( A ) A plasmid isolated from E . coli DC10B is transformed into staphylococci at 28°C, and single-crossover (SCO) integration was stimulated by growth at 37°C in the presence of chloramphenicol. The loss of replicating plasmid is assayed by colony PCR with MCS primers (IM151/IM152). Clones negative for replicating plasmid are then screened for the side of integration with a combination of chromosomal and cloning primers (e.g., OUT FWD/D REV [AB integration {AB INT}] or OUT REV/A FWD [CD integration {CD INT}]). The diagram details an integration event through the AB side (equivalent to clone h in panels B and C). A clone from either AB or CD integration event is grown at 28°C in broth without antibiotic selection to stimulate rolling circle replication and then plated on TSA with 1 µg/ml ATc. Expression of the secY antisense RNA ( a - secY ) inhibits growth of cells maintaining the plasmid. Plasmid excision through the AB side recreates the wild-type locus, while CD excision yields a mutated gene. (B) Colony PCR from 10 randomly chosen clones (clones a to j) after growth at 37°C for the presence of replicating plasmid. The absence of product indicates that the plasmid has integrated. Colony PCR from cells grown at 28°C is included as a positive control (+ve). (C) Two clones without replicating plasmid (clones c and h) were shown by colony PCR to have integrated either on the AB (upstream [clone h]) or CD (downstream [clone c]) side of the gene to be deleted. Wild-type (WT) genomic DNA was included as a control.

    Article Snippet: High-fidelity PCR was performed with KOD Hotstart DNA polymerase (Novagen) or Phusion DNA polymerase (Finnzymes) on genomic DNA isolated with the Genelute bacterial genomic DNA kit (Sigma).

    Techniques: Plasmid Preparation, Isolation, Transformation Assay, Polymerase Chain Reaction, Clone Assay, Selection, Expressing, Positive Control

    Adcy6 recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative PCR ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice

    doi: 10.1152/ajprenal.00397.2011

    Figure Lengend Snippet: Adcy6 recombination and AC6 mRNA expression in CD AC6 KO mice. A : representative PCR ( n = 6) of target organ recombination of Adcy6 gene. PCR was performed with primers spanning exons 2–13 of the mouse Adcy6 gene. The 0.5-kb band represents recombination

    Article Snippet: The adenylyl cyclase 6 gene ( Adcy6 ; ) fragment was obtained by high-fidelity PCR (Stratagene Pfu Ultra II Fusion HS DNA Polymerase, La Jolla, CA) of 129x1/SVJ genomic DNA (Jackson Laboratory, Bar Harbor, ME) using primers AC6-F 5′-AAC TGG TGA GAT GGC TCC TCA G-3′ and AC6-R 5′-GAC CCT CAG AAA ACA GAA GCG G-3′, adding Bam H1 to the upstream end and NOT 1 to the downstream end.

    Techniques: Expressing, Mouse Assay, Polymerase Chain Reaction