dna polymerase New England Biolabs Search Results


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  • 99
    New England Biolabs phusion hf dna polymerase new england biolabs
    Phusion Hf Dna Polymerase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna polymerase
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    New England Biolabs q5 high fidelity dna polymerase new england biolabs
    Q5 High Fidelity Dna Polymerase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs large klenow fragment new england biolabs
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    New England Biolabs phusion hot start polymerase new england biolabs
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    New England Biolabs q5 hot start high fidelty dna polymerase new england biolabs cat
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    New England Biolabs new england biolabs taq polymerase
    New England Biolabs Taq Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs recombinant proteins dnase i new england biolabs cat
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    New England Biolabs 3195l t4 dna ligase reaction buffer new england biolabs cat
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    New England Biolabs new england biolabs monarch pcr
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    New England Biolabs new england biolabs q5 high fidelity
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    New England Biolabs new england biolabs quick load taq
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    New England Biolabs m mulv reverse transcriptase new england biolabs
    M Mulv Reverse Transcriptase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs epimark 5 hmc
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    New England Biolabs t4 dna polymerase
    Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by <t>T4</t> DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phi29 dna polymerase
    Plasmid <t>DNA</t> from the cecal sample after amplification with <t>phi29</t> polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Phi29 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs vent dna polymerase
    Comparison of <t>NucC</t> binding to <t>DNA</t> fragments containing point mutations in the P2 P F activator binding site. Labeled DNA fragments of 154 bp were generated by PCR amplification from pFWT, pF51A, or pF64G as indicated. The triangles designate increasing NucC concentrations (0, 0.3, 0.6, 1.8, and 3.0 mM) in each set of five lanes. Complexes were resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.
    Vent Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 3965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs therminator dna polymerase
    TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a <t>DNA</t> template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, <t>Therminator,</t> 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.
    Therminator Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs φ29 dna polymerase
    ASF1 knockdown generates ECTR ( a ) Telomere Restriction Fragment (TRF) analysis of telomeric <t>DNA.</t> Left: Digested DNA (10µg) from independent prolonged ASF1 knockdowns (days 17 and 14 post transfection) in HeLa LT was resolved on a 0.7% TBE agarose gel. DNA from siControl transfected cells, Saos-2 and U2OS cells was resolved in parallel. Right: Lines traces of siControl (black) and siASF1 (red) gel lanes. ( b, c ) C-circle assays in siControl and siASF1 transfected IMR90-hTERT and HeLa LT. The ALT positive U2OS control is on the left. Negative controls are reactions lacking <t>φ29</t> or DNA. ( d ) Quantification of C-circles in siRNA and drug treated HeLa LT (light grey bar) and U2OS (dark grey bar). In both (c) and (d), C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value
    φ29 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by T4 DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P

    Journal: Genome Research

    Article Title: Guide Positioning Sequencing identifies aberrant DNA methylation patterns that alter cell identity and tumor-immune surveillance networks

    doi: 10.1101/gr.240606.118

    Figure Lengend Snippet: Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by T4 DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P

    Article Snippet: Thirty units of T4 DNA polymerase (New England BioLabs, M0203L) was used to perform 3′→5′ digestion of the DNA fragments for 100 min at 12°C followed by adding 10 µL dNTP mix which contained dATP, dTTP, dGTP, and 5′-methyl-dCTP nucleotide (final concentration 0.5 mM) and incubating for 30 min at 37°C.

    Techniques: Sequencing, Genome Wide, DNA Methylation Assay, Methylation, Next-Generation Sequencing

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Electroporation, Amplification

    Comparison of NucC binding to DNA fragments containing point mutations in the P2 P F activator binding site. Labeled DNA fragments of 154 bp were generated by PCR amplification from pFWT, pF51A, or pF64G as indicated. The triangles designate increasing NucC concentrations (0, 0.3, 0.6, 1.8, and 3.0 mM) in each set of five lanes. Complexes were resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Journal: Journal of Bacteriology

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

    doi: 10.1128/JB.185.6.1808-1816.2003

    Figure Lengend Snippet: Comparison of NucC binding to DNA fragments containing point mutations in the P2 P F activator binding site. Labeled DNA fragments of 154 bp were generated by PCR amplification from pFWT, pF51A, or pF64G as indicated. The triangles designate increasing NucC concentrations (0, 0.3, 0.6, 1.8, and 3.0 mM) in each set of five lanes. Complexes were resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Article Snippet: A 310-bp fragment containing the nucC gene was amplified from pSE380TacNucC by PCR with Vent DNA polymerase (New England Biolabs) and primers SM1 (5′-CGGAATTCT ATG ATGCACTGTCCACT-3′) and SM2 (5′-CACGTTGCATTTGCGAG-3′).

    Techniques: Binding Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Electrophoresis

    DNA bending analysis. (A) NucC electrophoretic mobility shift assays using the 153-bp DNA binding site fragments released from pBendF51 by digestion with Mlu I (a), Bgl II (b), Nhe I (c), Spe I (d), Eco RV (e), Pvu II (f), Stu I (g), Nru I (h), Kpn I (i), and Bam HI (j). (B) Plot of the relative mobility of NucC-DNA complexes as a function of the relative location of the NucC binding site within the 153-bp fragments. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Journal: Journal of Bacteriology

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

    doi: 10.1128/JB.185.6.1808-1816.2003

    Figure Lengend Snippet: DNA bending analysis. (A) NucC electrophoretic mobility shift assays using the 153-bp DNA binding site fragments released from pBendF51 by digestion with Mlu I (a), Bgl II (b), Nhe I (c), Spe I (d), Eco RV (e), Pvu II (f), Stu I (g), Nru I (h), Kpn I (i), and Bam HI (j). (B) Plot of the relative mobility of NucC-DNA complexes as a function of the relative location of the NucC binding site within the 153-bp fragments. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Article Snippet: A 310-bp fragment containing the nucC gene was amplified from pSE380TacNucC by PCR with Vent DNA polymerase (New England Biolabs) and primers SM1 (5′-CGGAATTCT ATG ATGCACTGTCCACT-3′) and SM2 (5′-CACGTTGCATTTGCGAG-3′).

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay

    Specificity of NucC activation in a coupled in vitro transcription-translation assay. The DNA template is indicated by the promoter fused to lacZ . Reaction mixtures 1 to 4 contained P4 sid promoter plasmid pSidZT (0.2 pmol), and reaction mixtures 5 to 8 contained lacUV5 promoter plasmid pRS229 (0.1 pmol). Purified NucC (30 pmol) was added as indicated to reaction mixtures 2 to 5. Competitor DNA (1 pmol, reaction mixtures 3 and 7, or 10 pmol, reaction mixtures 4 and 8) was a 154-bp fragment generated by PCR amplification of P2 P F promoter variant 64G 51A.

    Journal: Journal of Bacteriology

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

    doi: 10.1128/JB.185.6.1808-1816.2003

    Figure Lengend Snippet: Specificity of NucC activation in a coupled in vitro transcription-translation assay. The DNA template is indicated by the promoter fused to lacZ . Reaction mixtures 1 to 4 contained P4 sid promoter plasmid pSidZT (0.2 pmol), and reaction mixtures 5 to 8 contained lacUV5 promoter plasmid pRS229 (0.1 pmol). Purified NucC (30 pmol) was added as indicated to reaction mixtures 2 to 5. Competitor DNA (1 pmol, reaction mixtures 3 and 7, or 10 pmol, reaction mixtures 4 and 8) was a 154-bp fragment generated by PCR amplification of P2 P F promoter variant 64G 51A.

    Article Snippet: A 310-bp fragment containing the nucC gene was amplified from pSE380TacNucC by PCR with Vent DNA polymerase (New England Biolabs) and primers SM1 (5′-CGGAATTCT ATG ATGCACTGTCCACT-3′) and SM2 (5′-CACGTTGCATTTGCGAG-3′).

    Techniques: Activation Assay, In Vitro, Plasmid Preparation, Purification, Generated, Polymerase Chain Reaction, Amplification, Variant Assay

    Titration of DNA binding by NucC. The DNA template was a 154-bp P F fragment generated by PCR amplification from pFWT. Various amounts of purified NucC protein, as indicated, were incubated with approximately 1 ng of the labeled probe and resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel (19:1) containing 5% glycerol at 4°C for 2.5 h at 100 V. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Journal: Journal of Bacteriology

    Article Title: Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

    doi: 10.1128/JB.185.6.1808-1816.2003

    Figure Lengend Snippet: Titration of DNA binding by NucC. The DNA template was a 154-bp P F fragment generated by PCR amplification from pFWT. Various amounts of purified NucC protein, as indicated, were incubated with approximately 1 ng of the labeled probe and resolved by electrophoresis in 0.5× TBE on a 6% polyacrylamide gel (19:1) containing 5% glycerol at 4°C for 2.5 h at 100 V. This figure was compiled by using Adobe Photoshop and Microsoft PowerPoint.

    Article Snippet: A 310-bp fragment containing the nucC gene was amplified from pSE380TacNucC by PCR with Vent DNA polymerase (New England Biolabs) and primers SM1 (5′-CGGAATTCT ATG ATGCACTGTCCACT-3′) and SM2 (5′-CACGTTGCATTTGCGAG-3′).

    Techniques: Titration, Binding Assay, Generated, Polymerase Chain Reaction, Amplification, Purification, Incubation, Labeling, Electrophoresis

    PCR amplification of DNA fragments containing two Ds bases (60-, 62-, 65- or 68-mer). ( a ) Scheme for the PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. The amplified DNA fragments containing FAM-hx- Px were detected by denaturing PAGE. ( b ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on the gel was detected with an FLA-7000 bio-imager, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. ( c ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of NH 2 -hx-d Px TP and d Ds TP. ( d ) Sequencing of the 15-cycle amplified DNA fragments, in the presence of d Pa ′TP (50 μM). The PCR products amplified in the presence of NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds -strands. The arrows indicate the unnatural base position.

    Journal: Nucleic Acids Research

    Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    doi: 10.1093/nar/gkn956

    Figure Lengend Snippet: PCR amplification of DNA fragments containing two Ds bases (60-, 62-, 65- or 68-mer). ( a ) Scheme for the PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. The amplified DNA fragments containing FAM-hx- Px were detected by denaturing PAGE. ( b ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on the gel was detected with an FLA-7000 bio-imager, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. ( c ) Polyacrylamide-gel analysis of the PCR products amplified by 15 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of NH 2 -hx-d Px TP and d Ds TP. ( d ) Sequencing of the 15-cycle amplified DNA fragments, in the presence of d Pa ′TP (50 μM). The PCR products amplified in the presence of NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds -strands. The arrows indicate the unnatural base position.

    Article Snippet: After five rounds of in vitro selection, about 1 pmol of the recovered DNA was amplified by eight cycles of PCR (25 μl), in a reaction buffer with 50 μM NH2 -hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP, 1 μM each of 5′-primer (40-mer, 5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3′) and 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.02 U/μl DeepVent DNA polymerase, and then the PCR products were purified by electrophoresis on 8% polyacrylamide—7 M urea gel for direct sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Labeling, Fluorescence, Radioactivity, Autoradiography, Sequencing

    PCR amplification of DNA fragments (55-mer) containing one Ds base. ( a ) Scheme for PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. To detect DNA fragments containing FAM-hx- Px , the PCR products were analyzed by denaturing PAGE. DNA fragments amplified with NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds strands. ( b ) Polyacrylamide-gel analysis of the DNA fragments amplified by 15 or 30 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on a gel was detected with a bio-imaging analyzer, FLA-7000, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. The fold amplification of each DNA fragment is summarized in Table S3. ( c ) Sequencing of the 40-cycle amplified DNA fragments, in the presence of d Pa′ TP (2 μM) or dd Pa′ TP (50 μM). The arrows indicate the unnatural base position.

    Journal: Nucleic Acids Research

    Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    doi: 10.1093/nar/gkn956

    Figure Lengend Snippet: PCR amplification of DNA fragments (55-mer) containing one Ds base. ( a ) Scheme for PCR amplification in the presence of FAM-hx-d Px TP or NH 2 -hx-d Px TP with DeepVent DNA Pol. To detect DNA fragments containing FAM-hx- Px , the PCR products were analyzed by denaturing PAGE. DNA fragments amplified with NH 2 -hx-d Px TP and d Ds TP were used for sequencing of the Ds strands. ( b ) Polyacrylamide-gel analysis of the DNA fragments amplified by 15 or 30 cycles of PCR with the 32 P-labeled 5′-primer, in the presence of FAM-hx-d Px TP and d Ds TP. The fluorescence of the FAM-labeled full-length products on a gel was detected with a bio-imaging analyzer, FLA-7000, and the radioactivity of the full-length products on the same gel was analyzed by autoradiography. The fold amplification of each DNA fragment is summarized in Table S3. ( c ) Sequencing of the 40-cycle amplified DNA fragments, in the presence of d Pa′ TP (2 μM) or dd Pa′ TP (50 μM). The arrows indicate the unnatural base position.

    Article Snippet: After five rounds of in vitro selection, about 1 pmol of the recovered DNA was amplified by eight cycles of PCR (25 μl), in a reaction buffer with 50 μM NH2 -hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP, 1 μM each of 5′-primer (40-mer, 5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3′) and 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.02 U/μl DeepVent DNA polymerase, and then the PCR products were purified by electrophoresis on 8% polyacrylamide—7 M urea gel for direct sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Sequencing, Labeling, Fluorescence, Imaging, Radioactivity, Autoradiography

    Overview of in vitro selection using a Ds -containing DNA library. ( a ) Scheme for in vitro selection using the Ds -containing DNA library, by FAM-hx- Px incorporation into PCR products and isolation with an anti-fluorescein antibody. A chemically synthesized, single-stranded DNA library containing an NNN Ds NNN sequence (55-mer) was amplified by 10 cycles of PCR, in the presence of natural dNTPs, d Ds TP and FAM-hx-d Px TP, with DeepVent DNA Pol. After selection of the PCR products containing FAM-hx- Px by binding with an anti-fluorescein antibody, the isolated DNA fragments were used as a template for the next round of PCR amplification and selection. For direct sequencing of the library after five rounds of selection, the isolated DNA fragments were amplified in the presence of NH 2 -hx-d Px TP, instead of FAM-hx-d Px TP ( Figure 3 a), by eight cycles of PCR. Direct sequencing was performed in the presence of 2 μM d Pa′ TP, using a BigDye terminator v1.1 Cycle Sequencing kit. For the sequencing of clones after five rounds of selection, the library was amplified in the presence of only the natural dNTPs with Taq DNA polymerase, and the PCR products were used for TOPO TA cloning. ( b ) Sequencing of the DNA library after five rounds of selection. ( c ) Sequencing of the initial library. The arrow indicates the unnatural base position. ( d ) Probability (%) of occurrence at each position of the selected 66-clone sequences ( Supplementary Figure 1 ). Bases with an occurrence rate of ≥35% among the clones are colored red.

    Journal: Nucleic Acids Research

    Article Title: An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    doi: 10.1093/nar/gkn956

    Figure Lengend Snippet: Overview of in vitro selection using a Ds -containing DNA library. ( a ) Scheme for in vitro selection using the Ds -containing DNA library, by FAM-hx- Px incorporation into PCR products and isolation with an anti-fluorescein antibody. A chemically synthesized, single-stranded DNA library containing an NNN Ds NNN sequence (55-mer) was amplified by 10 cycles of PCR, in the presence of natural dNTPs, d Ds TP and FAM-hx-d Px TP, with DeepVent DNA Pol. After selection of the PCR products containing FAM-hx- Px by binding with an anti-fluorescein antibody, the isolated DNA fragments were used as a template for the next round of PCR amplification and selection. For direct sequencing of the library after five rounds of selection, the isolated DNA fragments were amplified in the presence of NH 2 -hx-d Px TP, instead of FAM-hx-d Px TP ( Figure 3 a), by eight cycles of PCR. Direct sequencing was performed in the presence of 2 μM d Pa′ TP, using a BigDye terminator v1.1 Cycle Sequencing kit. For the sequencing of clones after five rounds of selection, the library was amplified in the presence of only the natural dNTPs with Taq DNA polymerase, and the PCR products were used for TOPO TA cloning. ( b ) Sequencing of the DNA library after five rounds of selection. ( c ) Sequencing of the initial library. The arrow indicates the unnatural base position. ( d ) Probability (%) of occurrence at each position of the selected 66-clone sequences ( Supplementary Figure 1 ). Bases with an occurrence rate of ≥35% among the clones are colored red.

    Article Snippet: After five rounds of in vitro selection, about 1 pmol of the recovered DNA was amplified by eight cycles of PCR (25 μl), in a reaction buffer with 50 μM NH2 -hx-d Px TP, 50 μM d Ds TP, 0.3 mM each natural dNTP, 1 μM each of 5′-primer (40-mer, 5′-CGTTGTAAAACGACGGCCAGGATAATACGACTCACTATAG-3′) and 3′-primer (24-mer, 5′-TTTCACACAGGAAACAGCTATGAC-3′) and 0.02 U/μl DeepVent DNA polymerase, and then the PCR products were purified by electrophoresis on 8% polyacrylamide—7 M urea gel for direct sequencing.

    Techniques: In Vitro, Selection, Polymerase Chain Reaction, Isolation, Synthesized, Sequencing, Amplification, Binding Assay, Clone Assay, TA Cloning

    TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a DNA template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, Therminator, 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.

    Journal: Journal of the American Chemical Society

    Article Title: Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase

    doi: 10.1021/ja0428255

    Figure Lengend Snippet: TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a DNA template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, Therminator, 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.

    Article Snippet: Polymerization reactions were initiated by adding 10 μL of 2 × dNTP or tNTP solution (0.01−10 μM) to an equal volume of the reaction mixture containing primer−template complex, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM MgSO4 , 0.1% Triton X-100, 0.25 μg/μL BSA, 100 μM DTT, and either 0.05 units of Therminator DNA polymerase (final concentration 9.3 nM) (New England Biolabs, 2 u/μL) or 0.1 units of Deep Vent (exo-) DNA polymerase (final concentration 1.82 nM) (New England Biolabs, 2 u/μL).

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Mutagenesis

    TNA primer extension reactions. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for (A) Deep Vent exo- (DV exo- ) and (B) Therminator-catalyzed DNA polymerase reactions. Primer-extension reactions were performed in the absence (lanes 1−7) and presence (lanes 8−14) of 1.25 mM MnCl 2 .

    Journal: Journal of the American Chemical Society

    Article Title: Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase

    doi: 10.1021/ja0428255

    Figure Lengend Snippet: TNA primer extension reactions. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for (A) Deep Vent exo- (DV exo- ) and (B) Therminator-catalyzed DNA polymerase reactions. Primer-extension reactions were performed in the absence (lanes 1−7) and presence (lanes 8−14) of 1.25 mM MnCl 2 .

    Article Snippet: Polymerization reactions were initiated by adding 10 μL of 2 × dNTP or tNTP solution (0.01−10 μM) to an equal volume of the reaction mixture containing primer−template complex, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM MgSO4 , 0.1% Triton X-100, 0.25 μg/μL BSA, 100 μM DTT, and either 0.05 units of Therminator DNA polymerase (final concentration 9.3 nM) (New England Biolabs, 2 u/μL) or 0.1 units of Deep Vent (exo-) DNA polymerase (final concentration 1.82 nM) (New England Biolabs, 2 u/μL).

    Techniques: Polyacrylamide Gel Electrophoresis

    ASF1 knockdown generates ECTR ( a ) Telomere Restriction Fragment (TRF) analysis of telomeric DNA. Left: Digested DNA (10µg) from independent prolonged ASF1 knockdowns (days 17 and 14 post transfection) in HeLa LT was resolved on a 0.7% TBE agarose gel. DNA from siControl transfected cells, Saos-2 and U2OS cells was resolved in parallel. Right: Lines traces of siControl (black) and siASF1 (red) gel lanes. ( b, c ) C-circle assays in siControl and siASF1 transfected IMR90-hTERT and HeLa LT. The ALT positive U2OS control is on the left. Negative controls are reactions lacking φ29 or DNA. ( d ) Quantification of C-circles in siRNA and drug treated HeLa LT (light grey bar) and U2OS (dark grey bar). In both (c) and (d), C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value

    Journal: Nature structural & molecular biology

    Article Title: Rapid induction of Alternative Lengthening of Telomeres by depletion of the histone chaperone ASF1

    doi: 10.1038/nsmb.2754

    Figure Lengend Snippet: ASF1 knockdown generates ECTR ( a ) Telomere Restriction Fragment (TRF) analysis of telomeric DNA. Left: Digested DNA (10µg) from independent prolonged ASF1 knockdowns (days 17 and 14 post transfection) in HeLa LT was resolved on a 0.7% TBE agarose gel. DNA from siControl transfected cells, Saos-2 and U2OS cells was resolved in parallel. Right: Lines traces of siControl (black) and siASF1 (red) gel lanes. ( b, c ) C-circle assays in siControl and siASF1 transfected IMR90-hTERT and HeLa LT. The ALT positive U2OS control is on the left. Negative controls are reactions lacking φ29 or DNA. ( d ) Quantification of C-circles in siRNA and drug treated HeLa LT (light grey bar) and U2OS (dark grey bar). In both (c) and (d), C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value

    Article Snippet: Half (10 µl) of the annealed reaction was incubated at 30 °C for 12 h in the presence of dNTPs (0.2 mM each), BSA (NEB; 0.2 mg/ml), 1× Φ29 buffer (NEB) and 7.5U Φ29 DNA polymerase (NEB) in a total volume of 20 µl, and the remaining half was incubated under identical conditions with one exception; the Φ29 DNA polymerase was excluded from the reaction.

    Techniques: Transfection, Agarose Gel Electrophoresis