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  • 99
    Millipore dna fragment
    Nonradioactive EMSA of <t>Cra</t> and its binding site in aceBAK . EMSA experiment using 0.75 μM of the Cra site-containing <t>DNA</t> fragment and increasing concentrations of wild type Cra or the Cra mutant proteins in the presence of FBP ( a ) and the gray value of free DNA determined using Quantity one ( b ) and in the absence of FBP ( c ) and the gray value of free DNA determined using Quantity one ( d ). Lane 1, no protein; lane 2–5, 1, 2, 3, 4 μM Cra; lanes 6–9, 1, 2, 3, 4 μM Cra mutant (Tang1541); lanes 10–13, 1, 2, 3, 4 μM Cra mutant (Tang1544), respectively.
    Dna Fragment, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher nolimits 50 bp dna fragment
    Nonradioactive EMSA of <t>Cra</t> and its binding site in aceBAK . EMSA experiment using 0.75 μM of the Cra site-containing <t>DNA</t> fragment and increasing concentrations of wild type Cra or the Cra mutant proteins in the presence of FBP ( a ) and the gray value of free DNA determined using Quantity one ( b ) and in the absence of FBP ( c ) and the gray value of free DNA determined using Quantity one ( d ). Lane 1, no protein; lane 2–5, 1, 2, 3, 4 μM Cra; lanes 6–9, 1, 2, 3, 4 μM Cra mutant (Tang1541); lanes 10–13, 1, 2, 3, 4 μM Cra mutant (Tang1544), respectively.
    Nolimits 50 Bp Dna Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche deoxyribonucleic acid dna fragments
    Assessment of plasma membrane and <t>DNA</t> integrity. ( a ) Evaluation of sperm membrane integrity. Histograms show the incorporation of propidium iodide (PI) into spermatozoa from the caput and cauda epididymidis. ( b ) Evaluation of sperm DNA fragmentation from the Halomax detection assay, based on the sperm chromatin dispersion (SCD) test and the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and neutral comet assays (percentage of comet tail DNA). Values are expressed as mean±s.e.m. * P
    Deoxyribonucleic Acid Dna Fragments, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna fragment
    Intracellular expression of viral proteins of <t>prM</t> and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear <t>DNA</t> was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).
    Dna Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 548 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cosmo Genetech Co dna fragments
    Intracellular expression of viral proteins of <t>prM</t> and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear <t>DNA</t> was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).
    Dna Fragments, supplied by Cosmo Genetech Co, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novogene dna fragment
    Intracellular expression of viral proteins of <t>prM</t> and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear <t>DNA</t> was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).
    Dna Fragment, supplied by Novogene, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    ATUM dna fragments
    Intracellular expression of viral proteins of <t>prM</t> and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear <t>DNA</t> was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).
    Dna Fragments, supplied by ATUM, used in various techniques. Bioz Stars score: 96/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    CEM Corporation dna fragments
    Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear <t>DNA</t> cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two <t>CEM-T4</t> clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.
    Dna Fragments, supplied by CEM Corporation, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc dna fragments
    Preparation of paired-end reduced representation libraries. Genomic <t>DNA</t> is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large <t>contig</t> sequences.
    Dna Fragments, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 6938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Kapa Biosystems dna fragments
    Preparation of paired-end reduced representation libraries. Genomic <t>DNA</t> is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large <t>contig</t> sequences.
    Dna Fragments, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 94/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche dna fragments
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
    Dna Fragments, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 3706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    TaKaRa dna fragment
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
    Dna Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MWG-Biotech dna fragments
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
    Dna Fragments, supplied by MWG-Biotech, used in various techniques. Bioz Stars score: 93/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    APELEX dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by APELEX, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 5417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Genewiz dna fragment
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragment, supplied by Genewiz, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GenScript dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by GenScript, used in various techniques. Bioz Stars score: 99/100, based on 407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Twist Bioscience dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by Twist Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Blue Heron Biotech dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by Blue Heron Biotech, used in various techniques. Bioz Stars score: 84/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cambrex dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by Cambrex, used in various techniques. Bioz Stars score: 86/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dna fragments
    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were <t>PCR</t> amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual <t>DNA</t> fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.
    Dna Fragments, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 8246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Advanced Analytical Inc bp dna fragments
    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were <t>PCR</t> amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual <t>DNA</t> fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.
    Bp Dna Fragments, supplied by Advanced Analytical Inc, used in various techniques. Bioz Stars score: 84/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Advanced Analytical Inc dna fragment analyzer
    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were <t>PCR</t> amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual <t>DNA</t> fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.
    Dna Fragment Analyzer, supplied by Advanced Analytical Inc, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher short dna fragment
    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were <t>PCR</t> amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual <t>DNA</t> fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.
    Short Dna Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher string dna fragments
    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were <t>PCR</t> amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual <t>DNA</t> fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.
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    Thermo Fisher strings dna fragments
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Corbett Life Science dna fragment analyzer
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    TaKaRa dna fragments amplification
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Meridian Life Science esr1 dna fragment
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Sangon Biotech linker dna fragments
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Millipore oligonucleotide dna fragments
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Image Search Results


    Nonradioactive EMSA of Cra and its binding site in aceBAK . EMSA experiment using 0.75 μM of the Cra site-containing DNA fragment and increasing concentrations of wild type Cra or the Cra mutant proteins in the presence of FBP ( a ) and the gray value of free DNA determined using Quantity one ( b ) and in the absence of FBP ( c ) and the gray value of free DNA determined using Quantity one ( d ). Lane 1, no protein; lane 2–5, 1, 2, 3, 4 μM Cra; lanes 6–9, 1, 2, 3, 4 μM Cra mutant (Tang1541); lanes 10–13, 1, 2, 3, 4 μM Cra mutant (Tang1544), respectively.

    Journal: Scientific Reports

    Article Title: Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

    doi: 10.1038/srep36526

    Figure Lengend Snippet: Nonradioactive EMSA of Cra and its binding site in aceBAK . EMSA experiment using 0.75 μM of the Cra site-containing DNA fragment and increasing concentrations of wild type Cra or the Cra mutant proteins in the presence of FBP ( a ) and the gray value of free DNA determined using Quantity one ( b ) and in the absence of FBP ( c ) and the gray value of free DNA determined using Quantity one ( d ). Lane 1, no protein; lane 2–5, 1, 2, 3, 4 μM Cra; lanes 6–9, 1, 2, 3, 4 μM Cra mutant (Tang1541); lanes 10–13, 1, 2, 3, 4 μM Cra mutant (Tang1544), respectively.

    Article Snippet: Cra was incubated with the DNA fragment in the presence of 1 mM FBP (Sigma, 98% purity).

    Techniques: Binding Assay, Mutagenesis

    SNP analysis of centromeric domains. Sanger sequence traces from input ( above ) and CENP-A immunoprecipitated ( below ) samples from HSF-C, HSF-G, HSF-D and HSF-E. SNP coordinates are beneath traces. Stars indicate SNPs. For HSF-C, HSF-G, HSF-D-edge and HSF-E-edge, both nucleotides are present in input DNA while the immunoprecipitated DNA is enriched for one of the two nucleotides. For HSF-D centre and HSF-E centre, the two nucleotides are present in both input and CENP-A immunoprecipitated samples

    Journal: Chromosoma

    Article Title: Centromere sliding on a mammalian chromosome

    doi: 10.1007/s00412-014-0493-6

    Figure Lengend Snippet: SNP analysis of centromeric domains. Sanger sequence traces from input ( above ) and CENP-A immunoprecipitated ( below ) samples from HSF-C, HSF-G, HSF-D and HSF-E. SNP coordinates are beneath traces. Stars indicate SNPs. For HSF-C, HSF-G, HSF-D-edge and HSF-E-edge, both nucleotides are present in input DNA while the immunoprecipitated DNA is enriched for one of the two nucleotides. For HSF-D centre and HSF-E centre, the two nucleotides are present in both input and CENP-A immunoprecipitated samples

    Article Snippet: Both input and immunoprecipitated DNA fragments were purified and amplified using the whole genome amplification (WGA) kit (Sigma-Aldrich, St. Louis, USA).

    Techniques: Sequencing, Immunoprecipitation

    Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x -axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3 ). b Peak positions are represented as boxes . Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3 ) and fibre FISH (Fig. 4 and Supplementary Table 2 ) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser ( http://genome.ucsc.edu ). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4 ) and SNP analysis (Fig. 3 and Supplementary Table 2 ), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb

    Journal: Chromosoma

    Article Title: Centromere sliding on a mammalian chromosome

    doi: 10.1007/s00412-014-0493-6

    Figure Lengend Snippet: Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x -axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3 ). b Peak positions are represented as boxes . Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3 ) and fibre FISH (Fig. 4 and Supplementary Table 2 ) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser ( http://genome.ucsc.edu ). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4 ) and SNP analysis (Fig. 3 and Supplementary Table 2 ), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb

    Article Snippet: Both input and immunoprecipitated DNA fragments were purified and amplified using the whole genome amplification (WGA) kit (Sigma-Aldrich, St. Louis, USA).

    Techniques: Chromatin Immunoprecipitation, Hybridization, Immunoprecipitation, Fluorescence In Situ Hybridization, Sequencing, Binding Assay

    Whole genome amplification (WGA) as a method to generate double stranded DNA following DNA immunoprecipitation. ( a ) Schematic of the steps during the WGA. Red = WGA adapter sequences ( b ). Quantitative PCR (qPCR) analysis of several loci prior to WGA (post hmeDIP: light grey) as well as following WGA (dark grey) reveals little to no bias is introduced by the WGA. Analysis of the signals from the hmeDIP-SC-seq over these same regions highlights the retention of the patterns following sequencing (red bars). Loci were selected from previous work: 5hmC –ve = little/no 5hmC, 5hmC +ve = moderate/high 5hmC. Light grey bars: pre-amplified IP DNA, dark grey bars: post WGA DNA, red bars: normalised signals over locus used for qPCR taken from hmeDIP-SC-seq liver A dataset. qPCR data plotted against the primary y-axis, hmeDIP SC-seq data plotted against secondary y-axis ( c ). Glu-RES-qPCR results of three loci reveal quantitative information on 5hmC levels at a given site, confirming that pre and post WGA samples in fig. 2b reflect the expected 5hmC patterns. Tan: unmodified CpG, pink: methylated CpG, blue: hydroxymethylated CpG. ( d ) Examples of good and poor read length distributions following SC-seq. The majority of successfully sequenced DNA fragments should be > 100 bp with a mean around 125–150 bp. ( e ) Plot of mapping efficiency vs read length. Reads less than 50 bp should be excluded due to poor mapping accuracy.

    Journal: Scientific Reports

    Article Title: DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures

    doi: 10.1038/srep09778

    Figure Lengend Snippet: Whole genome amplification (WGA) as a method to generate double stranded DNA following DNA immunoprecipitation. ( a ) Schematic of the steps during the WGA. Red = WGA adapter sequences ( b ). Quantitative PCR (qPCR) analysis of several loci prior to WGA (post hmeDIP: light grey) as well as following WGA (dark grey) reveals little to no bias is introduced by the WGA. Analysis of the signals from the hmeDIP-SC-seq over these same regions highlights the retention of the patterns following sequencing (red bars). Loci were selected from previous work: 5hmC –ve = little/no 5hmC, 5hmC +ve = moderate/high 5hmC. Light grey bars: pre-amplified IP DNA, dark grey bars: post WGA DNA, red bars: normalised signals over locus used for qPCR taken from hmeDIP-SC-seq liver A dataset. qPCR data plotted against the primary y-axis, hmeDIP SC-seq data plotted against secondary y-axis ( c ). Glu-RES-qPCR results of three loci reveal quantitative information on 5hmC levels at a given site, confirming that pre and post WGA samples in fig. 2b reflect the expected 5hmC patterns. Tan: unmodified CpG, pink: methylated CpG, blue: hydroxymethylated CpG. ( d ) Examples of good and poor read length distributions following SC-seq. The majority of successfully sequenced DNA fragments should be > 100 bp with a mean around 125–150 bp. ( e ) Plot of mapping efficiency vs read length. Reads less than 50 bp should be excluded due to poor mapping accuracy.

    Article Snippet: This is achieved by converting the single stranded DNA fragments enriched by hmeDIP into double stranded fragments by carrying out a low number of whole genome amplification (WGA) cycles using a commercially available method optimised for next generational sequencing (Sigma Aldrich enhanced amplification kit) ( ).

    Techniques: Whole Genome Amplification, Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing, Amplification, Methylation

    In situ detection of DNA fragmentation in the Brachypodium callus cells on the 14th day of the culture using the TUNEL assay. Blue fluorescence: DAPI staining ( a – e ), green fluorescence: FITC marking TUNEL-positive nuclei ( a` – e` ). ( a , a` ) control callus; ( b , b` ) negative control in TUNEL reaction; ( c , c` ) positive control in TUNEL reaction; ( d , d` ) callus that has been treated with 5 μM of 5-azaC; ( e , e` ) callus that has been treated with 50 μM of 5-azaC. Scale bars, 50 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: 5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon

    doi: 10.3390/ijms19061806

    Figure Lengend Snippet: In situ detection of DNA fragmentation in the Brachypodium callus cells on the 14th day of the culture using the TUNEL assay. Blue fluorescence: DAPI staining ( a – e ), green fluorescence: FITC marking TUNEL-positive nuclei ( a` – e` ). ( a , a` ) control callus; ( b , b` ) negative control in TUNEL reaction; ( c , c` ) positive control in TUNEL reaction; ( d , d` ) callus that has been treated with 5 μM of 5-azaC; ( e , e` ) callus that has been treated with 50 μM of 5-azaC. Scale bars, 50 µm.

    Article Snippet: DNA fragment labelling was carried out using the TUNEL reaction mixture (In Situ Cell Death Detection Kit, Fluorescein, Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: In Situ, TUNEL Assay, Fluorescence, Staining, Negative Control, Positive Control

    Assessment of plasma membrane and DNA integrity. ( a ) Evaluation of sperm membrane integrity. Histograms show the incorporation of propidium iodide (PI) into spermatozoa from the caput and cauda epididymidis. ( b ) Evaluation of sperm DNA fragmentation from the Halomax detection assay, based on the sperm chromatin dispersion (SCD) test and the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and neutral comet assays (percentage of comet tail DNA). Values are expressed as mean±s.e.m. * P

    Journal: Asian Journal of Andrology

    Article Title: Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

    doi: 10.1038/aja.2011.119

    Figure Lengend Snippet: Assessment of plasma membrane and DNA integrity. ( a ) Evaluation of sperm membrane integrity. Histograms show the incorporation of propidium iodide (PI) into spermatozoa from the caput and cauda epididymidis. ( b ) Evaluation of sperm DNA fragmentation from the Halomax detection assay, based on the sperm chromatin dispersion (SCD) test and the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and neutral comet assays (percentage of comet tail DNA). Values are expressed as mean±s.e.m. * P

    Article Snippet: For the TUNEL assay, fragmented DNA was nick end-labelled with tetramethylrhodamine-conjugated dUTP by a terminal transferase ( In Situ Cell Death Detection Kit; Roche Molecular Biochemicals, Sant Cugat del Valles, Spain) for 1 h at 37 °C in the dark after chromatin denaturation according to the manufacturer's instructions.

    Techniques: Detection Assay, TUNEL Assay

    Intracellular expression of viral proteins of prM and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear DNA was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).

    Journal: Journal of Virology

    Article Title: Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly

    doi: 10.1128/JVI.00447-16

    Figure Lengend Snippet: Intracellular expression of viral proteins of prM and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear DNA was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).

    Article Snippet: The membrane anchor region ( and ) of wild-type (WT) TBEV E [E(T1-T2)] or prM [prM(T1-T2)] in the pTNd/c vector was replaced by a chemically synthesized DNA fragment (GeneArt AG, Germany) containing the heterologous membrane anchor of JEV [E(J1-J2) or prM(J1-J2)] or a shuffled membrane anchor [E(J1-T2), E(T1-J2), prM(J1-T2), or prM(T1-J2)] by using unique restriction sites.

    Techniques: Expressing, Immunofluorescence, Staining, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay

    DNA sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File S1 .

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Maize Transposable Elements Ac/Ds as Insertion Mutagenesis Tools in Candida albicans

    doi: 10.1534/g3.117.300388

    Figure Lengend Snippet: DNA sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File S1 .

    Article Snippet: Construction of Ac/Ds components The codon-adapted Ac TPase4xCa open reading frame (Supplemental Material, Figure S1 in File S1 ) was constructed by fusing three de novo -synthesized DNA fragments (Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany) and cloned into pJET1.2 via the CloneJET kit (Thermo Fisher Scientific).

    Techniques: Sequencing, Expressing

    DSB Repair and Targeted Genome Editing of the TCR Loci Using Designer Nucleases (A) During NHEJ-repair of TALEN- and CRISPR/Cas9-induced DSBs, frameshift mutations can result in gene knockout, or episomal IDLV can be integrated into DSBs, allowing for the permanent marking of off-target DSBs. If donor DNA is provided, HDR can lead to targeted integration of an expression cassette, i.e., therapeutic TCR chains. (B) The TCR α and β locus are composed of a number of variable (V), joining (J), constant (C), and, in the case of TRBC , diversity (D) gene segments (numbers of functional genes from IMGT/GENE-DB 53 version 3.1.16, December 14, 2016). The positions of TALEN and gRNA target sequences for TCR knockout in the TCR α constant region ( TRAC ) and a homologous sequence shared by both TCR β constant regions ( TRBC1/2 ) are marked by scissor symbols. DSB, DNA double-strand break; gRNA, guide RNA; HDR, homology-directed repair; IDLV, integrase-defective lentiviral vector; LTR, long terminal repeat; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; RVD, repeat variable di-residue.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

    doi: 10.1016/j.omtm.2017.01.005

    Figure Lengend Snippet: DSB Repair and Targeted Genome Editing of the TCR Loci Using Designer Nucleases (A) During NHEJ-repair of TALEN- and CRISPR/Cas9-induced DSBs, frameshift mutations can result in gene knockout, or episomal IDLV can be integrated into DSBs, allowing for the permanent marking of off-target DSBs. If donor DNA is provided, HDR can lead to targeted integration of an expression cassette, i.e., therapeutic TCR chains. (B) The TCR α and β locus are composed of a number of variable (V), joining (J), constant (C), and, in the case of TRBC , diversity (D) gene segments (numbers of functional genes from IMGT/GENE-DB 53 version 3.1.16, December 14, 2016). The positions of TALEN and gRNA target sequences for TCR knockout in the TCR α constant region ( TRAC ) and a homologous sequence shared by both TCR β constant regions ( TRBC1/2 ) are marked by scissor symbols. DSB, DNA double-strand break; gRNA, guide RNA; HDR, homology-directed repair; IDLV, integrase-defective lentiviral vector; LTR, long terminal repeat; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; RVD, repeat variable di-residue.

    Article Snippet: Donor Construction and Targeted Integration PCR For the 200 bp α chain donor construct (TA200G), we ordered a GeneArt Strings DNA Fragment (Thermo Fisher Scientific) containing two 200 bp regions flanking the αT4 cutting site in the TRAC locus and restriction sites AsiSI and SbfI for cloning into a lentiviral transfer vector in antisense direction.

    Techniques: Non-Homologous End Joining, CRISPR, Gene Knockout, Expressing, Functional Assay, Knock-Out, Sequencing, Plasmid Preparation

    Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear DNA cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two CEM-T4 clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.

    Journal: PLoS ONE

    Article Title: Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs

    doi: 10.1371/journal.pone.0127986

    Figure Lengend Snippet: Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear DNA cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two CEM-T4 clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.

    Article Snippet: Edited DNA fragments from CEM Clone 1 were isolated using primer sets flanking the provirus indicated in .

    Techniques: CRISPR, Mutagenesis, Knock-Out, Expressing, Flow Cytometry, Cytometry, shRNA, Negative Control, Viability Assay, Clone Assay, Plasmid Preparation, Transduction, Labeling, Sequencing

    Preparation of paired-end reduced representation libraries. Genomic DNA is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large contig sequences.

    Journal: International Journal of Plant Genomics

    Article Title: Local Assemblies of Paired-End Reduced Representation Libraries Sequenced with the Illumina Genome Analyzer in Maize

    doi: 10.1155/2012/360598

    Figure Lengend Snippet: Preparation of paired-end reduced representation libraries. Genomic DNA is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large contig sequences.

    Article Snippet: Contig length essentially varies with the size of the DNA fragments selected during the library construction procedure and is expected to be limited by the maximum length of the DNA fragments sequenced on the Illumina platform (separate experiments have shown that DNA fragments up to ~1 Kbps can be sequenced effectively on the Illumina Genome Analyzer).

    Techniques: Selection

    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated DIG-labeled DNA probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).

    Journal: Journal of Bacteriology

    Article Title: Dual Role of LldR in Regulation of the lldPRD Operon, Involved in l-Lactate Metabolism in Escherichia coli

    doi: 10.1128/JB.02013-07

    Figure Lengend Snippet: Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated DIG-labeled DNA probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).

    Article Snippet: A nonradioactive digoxigenin (DIG) gel shift kit for 3′-end labeling of DNA fragments (Roche Diagnostics, GmbH) was used for protein-DNA binding assays.

    Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Purification, Incubation, Polyacrylamide Gel Electrophoresis

    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with ethidium bromide for determination –108C > T polymorphism. DNA ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.

    Journal: The Open Biochemistry Journal

    Article Title: Alzheimer’s Disease and Paraoxonase 1 (PON1) Gene Polymorphisms

    doi: 10.2174/1874091X01711010047

    Figure Lengend Snippet: PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with ethidium bromide for determination –108C > T polymorphism. DNA ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.

    Article Snippet: Agarose gel (2%) stained with ethidium bromide (0.5 μg/ml) was used for electrophoresis of DNA fragments (Apelex, France).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were PCR amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual DNA fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.

    Journal: Journal of Bacteriology

    Article Title: Rex (Encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough Is a Repressor of Sulfate Adenylyl Transferase and Is Regulated by NADH

    doi: 10.1128/JB.02083-14

    Figure Lengend Snippet: EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were PCR amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual DNA fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.

    Article Snippet: Genomic DNA (Wizard Genomic DNA purification kit; Promega, Madison, WI), plasmid (GeneJET plasmid kit; Thermo Fisher Scientific), and DNA fragments (Wizard SV Gel and PCR Clean-Up System [Promega]) were purified according to the manufacturer's protocol.

    Techniques: Binding Assay, Polymerase Chain Reaction, Amplification, Generated, Purification, Concentration Assay, Labeling

    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with DNA encoding SthK-bP and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .

    Journal: Frontiers in Neuroscience

    Article Title: Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition

    doi: 10.3389/fnins.2018.00643

    Figure Lengend Snippet: Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with DNA encoding SthK-bP and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .

    Article Snippet: The SthK channel DNA sequence was synthesized by GeneArt Strings DNA Fragments (Life technologies, Thermo Fisher Scientific) according to the published amino acid sequence (Brams et al., ; Kesters et al., ) with codon usage optimized for Mus musculus .

    Techniques: Electroporation, Mass Spectrometry, Expressing, Generated, Injection