dna polymerase  (New England Biolabs)


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    DNA Polymerase I E coli
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    DNA Polymerase I E coli 2 500 units
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    DNA Polymerases
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    New England Biolabs dna polymerase
    DNA Polymerase I E coli
    DNA Polymerase I E coli 2 500 units
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    dna polymerase - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii"

    Article Title: Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii

    Journal: Genes

    doi: 10.3390/genes9110562

    Schematic diagram of the inverted-nested two-step PCR (INT-PCR, left) and the semi-random two-step PCR (ST-PCR, right) strategies to identify the transposon insertion sites in Haloferax volcanii . The transposable (Tn) element includes the following: two Mu repeats (MuR), a chloramphenicol acetyltransferase ( cat ) gene, a P cat promoter, a tryptophan synthase ( trpA ) gene, and a ferredoxin promoter (P fdx ). NdeI and HindIII are examples of the restriction enzyme (RE) sites used to cleave the genomic DNA prior to blunt-end ligation to form the circular DNA template used in the INT-PCR method. Primer pairs used for the two PCR steps (PCR1 and PCR2) and the DNA sequencing are color coded (red, blue, and purple) and numbered according to Table S1 . See Methods for details.
    Figure Legend Snippet: Schematic diagram of the inverted-nested two-step PCR (INT-PCR, left) and the semi-random two-step PCR (ST-PCR, right) strategies to identify the transposon insertion sites in Haloferax volcanii . The transposable (Tn) element includes the following: two Mu repeats (MuR), a chloramphenicol acetyltransferase ( cat ) gene, a P cat promoter, a tryptophan synthase ( trpA ) gene, and a ferredoxin promoter (P fdx ). NdeI and HindIII are examples of the restriction enzyme (RE) sites used to cleave the genomic DNA prior to blunt-end ligation to form the circular DNA template used in the INT-PCR method. Primer pairs used for the two PCR steps (PCR1 and PCR2) and the DNA sequencing are color coded (red, blue, and purple) and numbered according to Table S1 . See Methods for details.

    Techniques Used: Polymerase Chain Reaction, Ligation, DNA Sequencing

    2) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Role of conserved residue Y172 in the interaction with the DNAP. ( A ) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl 2 . ( B ) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. ( C ) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.
    Figure Legend Snippet: Role of conserved residue Y172 in the interaction with the DNAP. ( A ) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl 2 . ( B ) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. ( C ) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.

    Techniques Used: Mutagenesis, Sequencing, SDS Page

    Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.
    Figure Legend Snippet: Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.

    Techniques Used: Sequencing, Software, Incubation, SDS Page, Autoradiography, Electrophoresis, Expressing, Plasmid Preparation

    Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.
    Figure Legend Snippet: Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Marker

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    3) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.
    Figure Legend Snippet: Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.

    Techniques Used: Activity Assay, Radioactivity

    Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.
    Figure Legend Snippet: Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay

    4) Product Images from "Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins"

    Article Title: Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.00870-17

    Bam35 interactions found by different combinations of bait/prey fusion proteins. The numbers of reproducible protein-protein interactions (PPIs) detected with four, three, two, and one different combinations of vectors are indicated in red, green, blue, and black, respectively. The combination of vectors used, a graphic representation of the fusion protein obtained in each case, and the number of PPIs detected with each combination are indicated inside the boxes. AD, activation domain of the Gal4 transcription factor; DBD, DNA-binding domain of Gal4 transcription factor.
    Figure Legend Snippet: Bam35 interactions found by different combinations of bait/prey fusion proteins. The numbers of reproducible protein-protein interactions (PPIs) detected with four, three, two, and one different combinations of vectors are indicated in red, green, blue, and black, respectively. The combination of vectors used, a graphic representation of the fusion protein obtained in each case, and the number of PPIs detected with each combination are indicated inside the boxes. AD, activation domain of the Gal4 transcription factor; DBD, DNA-binding domain of Gal4 transcription factor.

    Techniques Used: Activation Assay, Binding Assay

    Genetic map of phage Bam35. The boxes correspond to the ORFs predicted to exist in the Bam35 genome. The ORFs that overlap another ORF(s) are represented at the bottom. Namely, the last 5 amino acids of ORF4 overlap ORF5, the last 8 amino acids of ORF7 overlap ORF8, the last 62 amino acids of ORF10 and the first 51 amino acids of ORF12 overlap ORF11, the last 10 amino acids of ORF12 and the first 6 amino acids of ORF14 overlap ORF13, the last amino acid of ORF21 and first amino acid of ORF24 overlap ORF23, the last 17 amino acids of ORF26 overlap ORF27, ORF31 is constituted by nucleotides 148 to 527 (in +1 frame) of ORF30, and ORF32 is constituted by the 102 C-terminal amino acids of ORF30. The previously shown or suggested (*) function of the protein or the type of protein encoded by an ORF is indicated as follows: green, DNA-binding protein; blue, DNA replication; chartreuse, cycle regulator; salmon, assembly or DNA packaging and other special vertex components; cyan, major capsid and spike proteins; purple, membrane structural protein; and orange, lytic proteins. Oblique lines indicate a predicted transmembrane domain. The ruler at the bottom represents the number of base pairs in the Bam35 genome. ITR, inverted terminal repeat; HVR, highly variable region. See Table S1 in the supplemental material for further details.
    Figure Legend Snippet: Genetic map of phage Bam35. The boxes correspond to the ORFs predicted to exist in the Bam35 genome. The ORFs that overlap another ORF(s) are represented at the bottom. Namely, the last 5 amino acids of ORF4 overlap ORF5, the last 8 amino acids of ORF7 overlap ORF8, the last 62 amino acids of ORF10 and the first 51 amino acids of ORF12 overlap ORF11, the last 10 amino acids of ORF12 and the first 6 amino acids of ORF14 overlap ORF13, the last amino acid of ORF21 and first amino acid of ORF24 overlap ORF23, the last 17 amino acids of ORF26 overlap ORF27, ORF31 is constituted by nucleotides 148 to 527 (in +1 frame) of ORF30, and ORF32 is constituted by the 102 C-terminal amino acids of ORF30. The previously shown or suggested (*) function of the protein or the type of protein encoded by an ORF is indicated as follows: green, DNA-binding protein; blue, DNA replication; chartreuse, cycle regulator; salmon, assembly or DNA packaging and other special vertex components; cyan, major capsid and spike proteins; purple, membrane structural protein; and orange, lytic proteins. Oblique lines indicate a predicted transmembrane domain. The ruler at the bottom represents the number of base pairs in the Bam35 genome. ITR, inverted terminal repeat; HVR, highly variable region. See Table S1 in the supplemental material for further details.

    Techniques Used: Binding Assay

    5) Product Images from "Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2"

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.013946-0

    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
    Figure Legend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Techniques Used: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    6) Product Images from "MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression"

    Article Title: MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002204

    The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218. ( A ) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). ( B ) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.
    Figure Legend Snippet: The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218. ( A ) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). ( B ) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Generated

    Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.
    Figure Legend Snippet: Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Generated, Purification, Incubation

    7) Product Images from "Effects of Vinylphosphonate Internucleotide Linkages on the Cleavage Specificity of Exonuclease III and on the Activity of DNA Polymerase I †"

    Article Title: Effects of Vinylphosphonate Internucleotide Linkages on the Cleavage Specificity of Exonuclease III and on the Activity of DNA Polymerase I †

    Journal: Biochemistry

    doi: 10.1021/bi026985+

    Primer extension reactions by DNA polymerase I (A) and Klenow (B). All reactions were carried out using DNA substrates P1 (unmodified) and P2 (modified) in the presence of dATP (lane 2), dATP and dGTP (lane 3), dATP, dGTP, and dCTP (lane 4), and dATP, dGTP, dCTP, and dTTP (lane 5). Lane 1 shows the radioactively labeled substrate, whereas the positions of the vinylphosphonate internucleotide linkages are marked with asterisks. Panel C shows similar reactions carried out with Klenow and DNA polymerase I but this time using DNA substrate P3 (one modification).
    Figure Legend Snippet: Primer extension reactions by DNA polymerase I (A) and Klenow (B). All reactions were carried out using DNA substrates P1 (unmodified) and P2 (modified) in the presence of dATP (lane 2), dATP and dGTP (lane 3), dATP, dGTP, and dCTP (lane 4), and dATP, dGTP, dCTP, and dTTP (lane 5). Lane 1 shows the radioactively labeled substrate, whereas the positions of the vinylphosphonate internucleotide linkages are marked with asterisks. Panel C shows similar reactions carried out with Klenow and DNA polymerase I but this time using DNA substrate P3 (one modification).

    Techniques Used: Modification, Labeling

    Synthetic oligonucleotides (A) and the DNA substrates (B) used in this study. The vinylphosphonate internucleotide linkages are denoted with asterisks, whereas the positions of the radioactive phosphate labels are denoted with bullets. Substrates N1–N4 and N7 were used for exonuclease III digestions and substrates N5 and N6 for mung bean nuclease digestions. Substrates P1–P3 were used for primer extension reactions catalyzed by Klenow and DNA polymerase I.
    Figure Legend Snippet: Synthetic oligonucleotides (A) and the DNA substrates (B) used in this study. The vinylphosphonate internucleotide linkages are denoted with asterisks, whereas the positions of the radioactive phosphate labels are denoted with bullets. Substrates N1–N4 and N7 were used for exonuclease III digestions and substrates N5 and N6 for mung bean nuclease digestions. Substrates P1–P3 were used for primer extension reactions catalyzed by Klenow and DNA polymerase I.

    Techniques Used:

    8) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    9) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    10) Product Images from "Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes"

    Article Title: Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagen.2009.11.003

    Metal response elements in Selh genes from nine species A. Position of MREs predicted in the Selh genes from nine species. Genomic DNA from the indicated species (dashed lines) was analyzed over the region spanning 1000bp upstream and downstream from the translational start (TrSS) sites, indicated by bent arrows below the dashed lines. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. Experimentally verified MTF-1 binding sites are marked by asterisks. MRE type a through f indicates the mouse metallothionein MRE with which the given MRE shows identity in the core sequence. MREx indicates that those sequences do not have a corresponding counterpart among the mouse metallothionein MREs. The conserved MREs located 200–350bp downstream of the TSS are encircled in the vertical box. Scale is shown at the lower left. B. Predicted MRE sequences in the human Selh gene. MRE1 through 4 predicted in the human Selh gene share identical core sequences (in bold) with MREs from the mouse metallothionein gene. MRE types are given in parentheses.
    Figure Legend Snippet: Metal response elements in Selh genes from nine species A. Position of MREs predicted in the Selh genes from nine species. Genomic DNA from the indicated species (dashed lines) was analyzed over the region spanning 1000bp upstream and downstream from the translational start (TrSS) sites, indicated by bent arrows below the dashed lines. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. Experimentally verified MTF-1 binding sites are marked by asterisks. MRE type a through f indicates the mouse metallothionein MRE with which the given MRE shows identity in the core sequence. MREx indicates that those sequences do not have a corresponding counterpart among the mouse metallothionein MREs. The conserved MREs located 200–350bp downstream of the TSS are encircled in the vertical box. Scale is shown at the lower left. B. Predicted MRE sequences in the human Selh gene. MRE1 through 4 predicted in the human Selh gene share identical core sequences (in bold) with MREs from the mouse metallothionein gene. MRE types are given in parentheses.

    Techniques Used: Binding Assay, Sequencing

    Mouse and human Selh genes are new targets of MTF-1 A. MTF-1 binding to the MRE-1 located in the 5′ UTR of the human Selh gene. ChIP assays were performed on MSTO-211H and HEK-293T cell extracts in the absence or presence of heavy metal treatment. Black bars represent immunoprecipitation with MTF-1 specific antibody (MTF-1 Ab) and white bars represent negative control (normal goat serum), indicated as no Ab. DNA fold enrichment units are normalized against input of total DNA used for immunoprecipitation and no Ab control. Three independent immunoprecipitations were carried out, immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. B. MTF-1 binding to MREs of mouse Selh coding region was assessed in MEF cells, as described in the legend for Fig 3A. Quantitation of MTF-1 binding for mouse MRE1 was evaluated by real time PCR (n=3) and data are plotted as mean ± SD. C . Gel electrophoresis analysis (4% polyacrylamide -TBE) of the amplification products from HEK-293T cell ChIP assays in the absence or presence Zn ++ addition. Band intensities from the gel were quantified using Adobe Photoshop software. Percent MTF-1 binding (signal minus background (no Ab) relative to input) is shown in the table below the gel pictures. Three independent immunoprecipitations were carried out and a representative of the output data is shown in this figure.
    Figure Legend Snippet: Mouse and human Selh genes are new targets of MTF-1 A. MTF-1 binding to the MRE-1 located in the 5′ UTR of the human Selh gene. ChIP assays were performed on MSTO-211H and HEK-293T cell extracts in the absence or presence of heavy metal treatment. Black bars represent immunoprecipitation with MTF-1 specific antibody (MTF-1 Ab) and white bars represent negative control (normal goat serum), indicated as no Ab. DNA fold enrichment units are normalized against input of total DNA used for immunoprecipitation and no Ab control. Three independent immunoprecipitations were carried out, immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. B. MTF-1 binding to MREs of mouse Selh coding region was assessed in MEF cells, as described in the legend for Fig 3A. Quantitation of MTF-1 binding for mouse MRE1 was evaluated by real time PCR (n=3) and data are plotted as mean ± SD. C . Gel electrophoresis analysis (4% polyacrylamide -TBE) of the amplification products from HEK-293T cell ChIP assays in the absence or presence Zn ++ addition. Band intensities from the gel were quantified using Adobe Photoshop software. Percent MTF-1 binding (signal minus background (no Ab) relative to input) is shown in the table below the gel pictures. Three independent immunoprecipitations were carried out and a representative of the output data is shown in this figure.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Quantitation Assay, Nucleic Acid Electrophoresis, Amplification, Software

    Mouse Txnrd2 gene is a new target of MTF-1 A. In-silico analysis of the mouse selenoprotein gene Txnrd 2 identified 3 putative MRE sequences. We analyzed the region spanning 1000bp upstream and downstream from the translational start site. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. MRE1 through 3 share identical core sequences (in bold) with MREs from the mouse metallothionein gene. The MRE types are given in parentheses. B. MTF-1 binding was assessed in MEF cells using primers specific for MRE2+3 in the absence or presence of heavy metal treatment as described in the text and in the legend for Fig. 3A. Three independent immunoprecipitations were carried. Immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. C. Mouse Txnrd2 mRNA expression was analyzed in MTF-1-KO and MTF-1FLAG cells in the absence of added metal. Three independent experiments were carried out. mRNAs levels relative to Hprt were analyzed by real time PCR ( n = 3) and are plotted as mean ± SD. Student’s t-test was used to evaluate statistical significance (indicated by asterisk) between the two groups.
    Figure Legend Snippet: Mouse Txnrd2 gene is a new target of MTF-1 A. In-silico analysis of the mouse selenoprotein gene Txnrd 2 identified 3 putative MRE sequences. We analyzed the region spanning 1000bp upstream and downstream from the translational start site. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. MRE1 through 3 share identical core sequences (in bold) with MREs from the mouse metallothionein gene. The MRE types are given in parentheses. B. MTF-1 binding was assessed in MEF cells using primers specific for MRE2+3 in the absence or presence of heavy metal treatment as described in the text and in the legend for Fig. 3A. Three independent immunoprecipitations were carried. Immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. C. Mouse Txnrd2 mRNA expression was analyzed in MTF-1-KO and MTF-1FLAG cells in the absence of added metal. Three independent experiments were carried out. mRNAs levels relative to Hprt were analyzed by real time PCR ( n = 3) and are plotted as mean ± SD. Student’s t-test was used to evaluate statistical significance (indicated by asterisk) between the two groups.

    Techniques Used: In Silico, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

    11) Product Images from "RAD54 controls access to the invading 3?-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae"

    Article Title: RAD54 controls access to the invading 3?-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn980

    Rad51–K191R requires higher Rad54 concentrations for efficient D-loop extension. ( A ) Rad51 titration in the D-loop extension assay with Pst I-95-mer: 0.17 µM (lanes 2–4), 0.34 µM (lanes 5–7), 0.5 µM (lanes 8–10), 0.67 µM (lanes 11–13), 1 µM (lanes 14–16), and 2 µM (lanes 18–20). Reactions contain 72 nM Rad54, as determined to be the optimum in Supplementary Figure 4 . Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker. ( B ) Rad51–K191R titration, otherwise as in (A). The signals labeled by asterisk in A and B are generated by the proofreading activity (3′–5′ exonuclease) of Klenow polymerase as determined in reconstruction experiments and verified using a proofreading-deficient version of Klenow polymerase (data not shown). This signal disappears when Rad51 fully occupies the 95-mer, further validating that Klenow polymerase has no access to the 3′-OH end with Rad51 bound to it. ( C ) Quantitation of the Rad51 and Rad51–K191R protein titration results in (a, b; 20 min time points). A higher stoichiometry (1/2 nt) is optimal for the Rad51–K191R protein compared to the 1/4 nt stoichiometry for the wild-type Rad51 protein. This was expected from previous results showing a DNA-binding defect for the Rad51–K191R protein, requiring higher protein to DNA ratios to assemble saturated protein filaments ( 7 ). ( D ) Titration of Rad54 in D-loop extension assay with Rad51 at optimal 1/4 stoichiometry. ( E ) Titration of Rad54 in D-loop extension assay with Rad51–K191R at optimal 1/2 stoichiometry. ( F ) Quantitation of results in (D, E; 20 min time points). The results were normalized for the amount of linear D-loops captured by psoralen crosslinking under each assay condition. D-loop extension with wild-type Rad51 reaches an optimum at 36 nM Rad54, whereas the optimum with Rad51–K191R is reached at 54 nM. Shown are the means from three determinations; error bars represent 1 SD.
    Figure Legend Snippet: Rad51–K191R requires higher Rad54 concentrations for efficient D-loop extension. ( A ) Rad51 titration in the D-loop extension assay with Pst I-95-mer: 0.17 µM (lanes 2–4), 0.34 µM (lanes 5–7), 0.5 µM (lanes 8–10), 0.67 µM (lanes 11–13), 1 µM (lanes 14–16), and 2 µM (lanes 18–20). Reactions contain 72 nM Rad54, as determined to be the optimum in Supplementary Figure 4 . Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker. ( B ) Rad51–K191R titration, otherwise as in (A). The signals labeled by asterisk in A and B are generated by the proofreading activity (3′–5′ exonuclease) of Klenow polymerase as determined in reconstruction experiments and verified using a proofreading-deficient version of Klenow polymerase (data not shown). This signal disappears when Rad51 fully occupies the 95-mer, further validating that Klenow polymerase has no access to the 3′-OH end with Rad51 bound to it. ( C ) Quantitation of the Rad51 and Rad51–K191R protein titration results in (a, b; 20 min time points). A higher stoichiometry (1/2 nt) is optimal for the Rad51–K191R protein compared to the 1/4 nt stoichiometry for the wild-type Rad51 protein. This was expected from previous results showing a DNA-binding defect for the Rad51–K191R protein, requiring higher protein to DNA ratios to assemble saturated protein filaments ( 7 ). ( D ) Titration of Rad54 in D-loop extension assay with Rad51 at optimal 1/4 stoichiometry. ( E ) Titration of Rad54 in D-loop extension assay with Rad51–K191R at optimal 1/2 stoichiometry. ( F ) Quantitation of results in (D, E; 20 min time points). The results were normalized for the amount of linear D-loops captured by psoralen crosslinking under each assay condition. D-loop extension with wild-type Rad51 reaches an optimum at 36 nM Rad54, whereas the optimum with Rad51–K191R is reached at 54 nM. Shown are the means from three determinations; error bars represent 1 SD.

    Techniques Used: Titration, Labeling, Marker, Generated, Activity Assay, Quantitation Assay, Binding Assay

    Rad54 is required for D-loop extension. D-loop extension assays. The Pst I-95-mer is homologous to the terminal sequence of the Pst I-linearized pUC19 DNA (2686 bp). Reaction products are identified either by end-labeling the 95-mer ( A , B , F ) or by incorporation of α- 32 P-dGTP ( C – E , G ). (A) D-loop extension assay with end-labeled Pst I-95-mer. Rad51 nucleoprotein filaments were incubated either in the presence of Rad54 (72 nM, lanes 9–11), or absence of Rad54 (lanes 6–8), or absence of DNA polymerase I (Klenow fragment 24 nM, lanes 12–14). Protein-free 95-mer was also incubated either in the presence of Rad54 (72 nM, lanes 3–5), or absence of Rad54 (lanes 15–17). Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker, and lane 2 the end-labeled 95-mer. (B) Quantification of the results for D-loop extension in (A). (C) D-loop extension assay with α- 32 P-dGTP and unlabeled Pst I-95-mer. Reactions were as in (A), except lane 2 contains unlabeled Pst I-95-mer and lanes 18–20 show Pst I-linearized pUC19 with DNA polymerase I (Klenow fragment, 24 nM). (D) Quantification of the stable extension product from (C). Stable extension products are D-loops of sufficient length to be stable under electrophoresis conditions. (E) Quantification of the unstable extension product from (C). Unstable extension products are extended 95-mers with insufficient length to result in stable D-loops under electrophoresis conditions. For (B)–(E) shown are means from three determinations; error bars represent 1 SD. (F) Analysis of extension products on denaturing gel from reactions with end-labeled 95-mer and (G) with α- 32 P-dGTP. The signals labeled by asterisk are due to a combination of 3′–5′ exonuclease (proofreading) and/or polymerase activity of Klenow polymerase on the 95-mer or the linear dsDNA.
    Figure Legend Snippet: Rad54 is required for D-loop extension. D-loop extension assays. The Pst I-95-mer is homologous to the terminal sequence of the Pst I-linearized pUC19 DNA (2686 bp). Reaction products are identified either by end-labeling the 95-mer ( A , B , F ) or by incorporation of α- 32 P-dGTP ( C – E , G ). (A) D-loop extension assay with end-labeled Pst I-95-mer. Rad51 nucleoprotein filaments were incubated either in the presence of Rad54 (72 nM, lanes 9–11), or absence of Rad54 (lanes 6–8), or absence of DNA polymerase I (Klenow fragment 24 nM, lanes 12–14). Protein-free 95-mer was also incubated either in the presence of Rad54 (72 nM, lanes 3–5), or absence of Rad54 (lanes 15–17). Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker, and lane 2 the end-labeled 95-mer. (B) Quantification of the results for D-loop extension in (A). (C) D-loop extension assay with α- 32 P-dGTP and unlabeled Pst I-95-mer. Reactions were as in (A), except lane 2 contains unlabeled Pst I-95-mer and lanes 18–20 show Pst I-linearized pUC19 with DNA polymerase I (Klenow fragment, 24 nM). (D) Quantification of the stable extension product from (C). Stable extension products are D-loops of sufficient length to be stable under electrophoresis conditions. (E) Quantification of the unstable extension product from (C). Unstable extension products are extended 95-mers with insufficient length to result in stable D-loops under electrophoresis conditions. For (B)–(E) shown are means from three determinations; error bars represent 1 SD. (F) Analysis of extension products on denaturing gel from reactions with end-labeled 95-mer and (G) with α- 32 P-dGTP. The signals labeled by asterisk are due to a combination of 3′–5′ exonuclease (proofreading) and/or polymerase activity of Klenow polymerase on the 95-mer or the linear dsDNA.

    Techniques Used: Sequencing, End Labeling, Labeling, Incubation, Marker, Electrophoresis, Activity Assay

    12) Product Images from "The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5?-diphosphate-ribose) polymerase-1"

    Article Title: The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5?-diphosphate-ribose) polymerase-1

    Journal: Blood

    doi: 10.1182/blood-2008-12-195768

    DNA repair by PARP-1 is disrupted by GzmA. (A) GzmA-induced DNA nicks, radiolabeled with Klenow, in HeLa cells are enhanced by inhibiting PARP-1 using the chemical inhibitor 1,5 dihydroxyisoquinoline (DIQ) and reduced by overexpressing WT PARP-1. (B)
    Figure Legend Snippet: DNA repair by PARP-1 is disrupted by GzmA. (A) GzmA-induced DNA nicks, radiolabeled with Klenow, in HeLa cells are enhanced by inhibiting PARP-1 using the chemical inhibitor 1,5 dihydroxyisoquinoline (DIQ) and reduced by overexpressing WT PARP-1. (B)

    Techniques Used:

    13) Product Images from "Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay"

    Article Title: Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01372

    Optimal reaction temperatures of LAMP. (A) Detecting LAMP products by adding fluorescence metal indicator (calcein); the assessment was based on visualization of a color change from brown to yellowish-green. (B) Agarose gel electrophoresis analysis of the LAMP products. In (A,B) , lane 1: 61°C, lane 2: 62°C, lane 3: 63°C, lane 4: 64°C, lane 5: 65°C, lane 6: 66°C, lane 7: 68°C. M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in all three replicates.
    Figure Legend Snippet: Optimal reaction temperatures of LAMP. (A) Detecting LAMP products by adding fluorescence metal indicator (calcein); the assessment was based on visualization of a color change from brown to yellowish-green. (B) Agarose gel electrophoresis analysis of the LAMP products. In (A,B) , lane 1: 61°C, lane 2: 62°C, lane 3: 63°C, lane 4: 64°C, lane 5: 65°C, lane 6: 66°C, lane 7: 68°C. M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in all three replicates.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Marker

    LAMP detection of D. bryoniae (DBJSJY2). Assessment is based on (A) LAMP for detection of D. bryoniae was using a fluorescence metal indicator (calcein) as a visual indicator. The positive reaction becomes yellowish-green, and the negative is still brown; (B) LAMP product was manifested as a ladder-like pattern on a 2.0% agarose gel. M: Trans DNA Marker II (Transgen Biotech, Beijing). In (A,B) , 1: Negative reaction (without target DNA), 2: Positive reaction (with target DNA). The same results were obtained in all three replicates.
    Figure Legend Snippet: LAMP detection of D. bryoniae (DBJSJY2). Assessment is based on (A) LAMP for detection of D. bryoniae was using a fluorescence metal indicator (calcein) as a visual indicator. The positive reaction becomes yellowish-green, and the negative is still brown; (B) LAMP product was manifested as a ladder-like pattern on a 2.0% agarose gel. M: Trans DNA Marker II (Transgen Biotech, Beijing). In (A,B) , 1: Negative reaction (without target DNA), 2: Positive reaction (with target DNA). The same results were obtained in all three replicates.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Marker

    Specificity of LAMP detection of D. bryoniae . Assessment was based on (A) fluorescence metal indicator calcein visualization of color change, (B) the turbidity analysis of the LAMP products or (C) agarose gel electrophoresis analysis of the LAMP products. Lane 1, Didymella bryoniae (strain DBJSJY2) RGI; lane 2, Didymella bryoniae (strain DBAHHF2,) RGI; lane 3, Didymella bryoniae (strain DBZJNB5) RGI; lane 4, Didymella bryoniae (strain DBJSNJ60) RGI; lane 5, Didymella bryoniae (strain DBZJNB7) RGII; lane 6, Ascochyta pinodes ZJ-1; lane 7, Colletotrichum orbiculare NJ-1; lane 8, Pythium paroecandrum Drechsler ; lane 9, Alternaria alternata LH1401; lane 10, Fusarium verticillioide ; lane 11, Fusarium oxysporum f.sp. niveum Race 0; lane 12, Fusarium oxysporum f.sp. niveum Race 1; lane 13, Fusarium oxysporum f.sp. niveum Race 2; lane 14, positive control; lane 15, negative control. M, Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Specificity of LAMP detection of D. bryoniae . Assessment was based on (A) fluorescence metal indicator calcein visualization of color change, (B) the turbidity analysis of the LAMP products or (C) agarose gel electrophoresis analysis of the LAMP products. Lane 1, Didymella bryoniae (strain DBJSJY2) RGI; lane 2, Didymella bryoniae (strain DBAHHF2,) RGI; lane 3, Didymella bryoniae (strain DBZJNB5) RGI; lane 4, Didymella bryoniae (strain DBJSNJ60) RGI; lane 5, Didymella bryoniae (strain DBZJNB7) RGII; lane 6, Ascochyta pinodes ZJ-1; lane 7, Colletotrichum orbiculare NJ-1; lane 8, Pythium paroecandrum Drechsler ; lane 9, Alternaria alternata LH1401; lane 10, Fusarium verticillioide ; lane 11, Fusarium oxysporum f.sp. niveum Race 0; lane 12, Fusarium oxysporum f.sp. niveum Race 1; lane 13, Fusarium oxysporum f.sp. niveum Race 2; lane 14, positive control; lane 15, negative control. M, Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Positive Control, Negative Control, Marker

    Optimal reaction time of LAMP. (A) Agarose gel electrophoresis analysis of the LAMP products. (B) Detecting LAMP products by adding a fluorescence metal indicators (calcein). In (A,B) , lane 1: 60 min, lane 2: 45 min, lane 3: 30 min, lane 4: 15 min, M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Optimal reaction time of LAMP. (A) Agarose gel electrophoresis analysis of the LAMP products. (B) Detecting LAMP products by adding a fluorescence metal indicators (calcein). In (A,B) , lane 1: 60 min, lane 2: 45 min, lane 3: 30 min, lane 4: 15 min, M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Agarose Gel Electrophoresis, Fluorescence, Marker

    Sensitivity of the LAMP and conventional PCR. LAMP and conventional PCR assays using 10-fold serial dilutions of purified target DNA from D. bryoniae genomic DNA (strain DBJSJY2). (A) Detecting LAMP products by adding a fluorescence metal indicator (calcein). (B) Agarose gel electrophoresis analysis of the LAMP products. (C) Conventional PCR. Concentrations of template DNA (fg μL -1 ) per reaction in (A,B) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1, lane 7 = 10 -1 and lane 8 = 10 -2 . Concentrations of template DNA (fg μL -1 ) per reaction in (C) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1 and lane 7 = 10 -1 . In (B,C) , M indicates a Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Sensitivity of the LAMP and conventional PCR. LAMP and conventional PCR assays using 10-fold serial dilutions of purified target DNA from D. bryoniae genomic DNA (strain DBJSJY2). (A) Detecting LAMP products by adding a fluorescence metal indicator (calcein). (B) Agarose gel electrophoresis analysis of the LAMP products. (C) Conventional PCR. Concentrations of template DNA (fg μL -1 ) per reaction in (A,B) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1, lane 7 = 10 -1 and lane 8 = 10 -2 . Concentrations of template DNA (fg μL -1 ) per reaction in (C) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1 and lane 7 = 10 -1 . In (B,C) , M indicates a Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Polymerase Chain Reaction, Purification, Fluorescence, Agarose Gel Electrophoresis, Marker

    14) Product Images from "Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein *"

    Article Title: Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.223495

    DnaA(L366K) in either nucleotide form represses in vitro transcription from dnaA promoter similar to ATP-DnaA. The indicated amounts of ATP and ADP forms of DnaA(WT) ( A ) and DnaA(L366K) ( B ) were incubated with the template DNA, and RNA synthesis was initiated
    Figure Legend Snippet: DnaA(L366K) in either nucleotide form represses in vitro transcription from dnaA promoter similar to ATP-DnaA. The indicated amounts of ATP and ADP forms of DnaA(WT) ( A ) and DnaA(L366K) ( B ) were incubated with the template DNA, and RNA synthesis was initiated

    Techniques Used: In Vitro, Incubation

    Related Articles

    Incubation:

    Article Title: LSH catalyzes ATP-driven exchange of histone variants macroH2A1 and macroH2A2
    Article Snippet: .. To generate biotinylated DNA fragment, 10 μg DNA was incubated with 7.5 μl NEBuffer 2 (10×), 2 μl of 0.4 mM biotin-14-dATP (Invitrogen), 2 μl of dBTP mix (1 mM dGTP, 1 mM dCTP, 1 mM dTTP), 1 μl of DNA polI (Klenow, NEB), 5 mM MgCl2 , 1.5 mM DTT and add H2O to the volume of 75 μl with incubation at room temperature for 30 min. Add 1.5 μl of 500 mM EDTA and incubate at 75°C for 20 min to stop reaction. ..

    Amplification:

    Article Title: The SARS-CoV-2 Programmed −1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography
    Article Snippet: .. Polyadenylated RNA was used as the template in a reverse transcription reaction using SuperScript III (Invitrogen) according to the manufacturer’s instructions. cDNA was amplified using end specific primers and PCR with 30 cycles of amplification using an annealing temperature of 52 °C andTaq DNA polymerase (NEB). ..

    Polymerase Chain Reaction:

    Article Title: The SARS-CoV-2 Programmed −1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography
    Article Snippet: .. Polyadenylated RNA was used as the template in a reverse transcription reaction using SuperScript III (Invitrogen) according to the manufacturer’s instructions. cDNA was amplified using end specific primers and PCR with 30 cycles of amplification using an annealing temperature of 52 °C andTaq DNA polymerase (NEB). ..

    Article Title: Molecular detection and genetic diversity of Leucocytozoon sabrazesi in chickens in Thailand
    Article Snippet: .. PCR reaction mixtures consisting of 50 ng DNA template, 0.2 µM each of the primers, 0.125 mM of each deoxynucleoside triphosphate (dNTPs), 3 mM MgCl2 , 0.25 U Tag DNA polymerase (NEW ENGLAND BIOLABS, UK), 1× standard Tag reaction buffer and nuclease free water, were put through in a thermal cycle (BIO-RAD, USA) with the following condition: 40 cycles of denaturation at 94 °C for 1 min (1st step) and for 20 s (2nd step), annealing at 50 °C for 1 min (1st step) and 53 °C for 20 s (2nd step), extension at 68 °C for 1 min (1st step) and for 30 s (2nd step) as well as a final extension at 68 °C for 5 min (1st and 2nd steps). ..

    Sequencing:

    Article Title: Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection
    Article Snippet: .. The resultant sequence corresponds to a T. th DNA polymerase I fragment, orthologous to the Stoffel fragment of T. aq polymerase, with a replacement in the finger domain derived from T. in . ..

    Derivative Assay:

    Article Title: Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection
    Article Snippet: .. The resultant sequence corresponds to a T. th DNA polymerase I fragment, orthologous to the Stoffel fragment of T. aq polymerase, with a replacement in the finger domain derived from T. in . ..

    Purification:

    Article Title: The Plant Salicylic Acid Signalling Pathway Regulates the Infection of a Biotrophic Pathogen in Grasses Associated with an Epichloë Endophyte
    Article Snippet: .. The mRNA was purified from 3 μg of total RNA using poly-T oligo-attached magnetic beads. cDNA strands were synthesised using random hexamer primers, M-MuLV reverse transcriptase (RNase H-) and DNA Polymerase I. NEBNext adaptors were ligated to double-stranded cDNA fragments. ..

    Magnetic Beads:

    Article Title: The Plant Salicylic Acid Signalling Pathway Regulates the Infection of a Biotrophic Pathogen in Grasses Associated with an Epichloë Endophyte
    Article Snippet: .. The mRNA was purified from 3 μg of total RNA using poly-T oligo-attached magnetic beads. cDNA strands were synthesised using random hexamer primers, M-MuLV reverse transcriptase (RNase H-) and DNA Polymerase I. NEBNext adaptors were ligated to double-stranded cDNA fragments. ..

    Random Hexamer Labeling:

    Article Title: The Plant Salicylic Acid Signalling Pathway Regulates the Infection of a Biotrophic Pathogen in Grasses Associated with an Epichloë Endophyte
    Article Snippet: .. The mRNA was purified from 3 μg of total RNA using poly-T oligo-attached magnetic beads. cDNA strands were synthesised using random hexamer primers, M-MuLV reverse transcriptase (RNase H-) and DNA Polymerase I. NEBNext adaptors were ligated to double-stranded cDNA fragments. ..

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  • 99
    New England Biolabs t7 endonuclease i
    Deletion of MCM10 through CRISPR/Cas9 technology. Notes:  ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P
    T7 Endonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase i klenow fragment
    Inhibition of Pol I results in <t>DNA</t> damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P
    Dna Polymerase I Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs dna polymerase i
    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of <t>DNA</t> <t>polymerase</t> I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.
    Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs klenow fragment exo buffer
    Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), <t>Klenow</t> Fragment <t>exo-</t> (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.
    Klenow Fragment Exo Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Deletion of MCM10 through CRISPR/Cas9 technology. Notes:  ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: Ablation of MCM10 using CRISPR/Cas9 restrains the growth and migration of esophageal squamous cell carcinoma cells through inhibition of Akt signaling

    doi: 10.2147/OTT.S157025

    Figure Lengend Snippet: Deletion of MCM10 through CRISPR/Cas9 technology. Notes: ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

    Article Snippet: After treatment with T7 endonuclease I (New England Biolabs, Ipswich, MA, USA) at 37°C for 2 h, the resulting fragments were subjected to 1% agarose gel electrophoresis and stained with ethidium bromide.

    Techniques: CRISPR, Knock-Out, Construct, Transfection, Fluorescence, Sequencing, Clone Assay, Derivative Assay, Polymerase Chain Reaction, Amplification, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Migration, In Vitro, Wound Healing Assay

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Journal: Nature

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    doi: 10.1038/nature25449

    Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

    Techniques: Inhibition, Immunofluorescence, Staining

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Article Snippet: Primer extension with Klenow fragment of DNA polymerase Primer extension experiments using large fragment (Klenow) of DNA polymerase I (New England Biolabs) were performed at 25°C using the HIV-1 tat reverse primer (5′-AATACTATGGTCCACACAACTATTGCT-3′) that was unmodified or contained a single OXP modification.

    Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), Klenow Fragment exo- (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins

    doi: 10.3390/s19010077

    Figure Lengend Snippet: Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), Klenow Fragment exo- (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.

    Article Snippet: The deoxynucleotide solution mixture (dNTPs), polymerase Klenow Fragment exo- (10 U/μL) accompanied by 10× Klenow Fragment exo- buffer, and the nicking endonuclease Nt.BbvCI accompanied by 10× New England Biolabs (NEB) buffer were purchased from New England Biolabs Ltd (Beijing, China).

    Techniques: Fluorescence

    The fluorescence spectrum of the developed sensing system is collected in a blank control sample (curve “a”); in the presence of aptamer DNA, an MB, polymerase, and nicking endonuclease, but without PDGF-BB (curve “b”); in the presence of PDGF-BB, aptamer DNA, and an MB (curve “c”); in the presence of PDGF-BB, aptamer DNA, an MB, and polymerase (curve “d”); and in the presence of PDGF-BB, aptamer DNA, MB, polymerase, and nicking endonuclease Nt.BbvCI (curve “e”). The reaction is in an NEB buffer (pH 7.9) at 37 °C for 2 h containing 1 μM aptamer DNA, 1 μM MB, 2.5 μM primer, 100 ng/mL PDGF-BB, 1 U/μL Klenow Fragment exo-, 2 mM dNTPs, and 1 U/μL Nt.BbvCI.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins

    doi: 10.3390/s19010077

    Figure Lengend Snippet: The fluorescence spectrum of the developed sensing system is collected in a blank control sample (curve “a”); in the presence of aptamer DNA, an MB, polymerase, and nicking endonuclease, but without PDGF-BB (curve “b”); in the presence of PDGF-BB, aptamer DNA, and an MB (curve “c”); in the presence of PDGF-BB, aptamer DNA, an MB, and polymerase (curve “d”); and in the presence of PDGF-BB, aptamer DNA, MB, polymerase, and nicking endonuclease Nt.BbvCI (curve “e”). The reaction is in an NEB buffer (pH 7.9) at 37 °C for 2 h containing 1 μM aptamer DNA, 1 μM MB, 2.5 μM primer, 100 ng/mL PDGF-BB, 1 U/μL Klenow Fragment exo-, 2 mM dNTPs, and 1 U/μL Nt.BbvCI.

    Article Snippet: The deoxynucleotide solution mixture (dNTPs), polymerase Klenow Fragment exo- (10 U/μL) accompanied by 10× Klenow Fragment exo- buffer, and the nicking endonuclease Nt.BbvCI accompanied by 10× New England Biolabs (NEB) buffer were purchased from New England Biolabs Ltd (Beijing, China).

    Techniques: Fluorescence