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Roche dna polymerase buffer
Dna Polymerase Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Incubation:

Article Title: DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells
Article Snippet: .. Baker) DNA ladder (25 bp) (Invitrogen) DNA Polymerase Buffer (1X; 10 mM Tris-Cl, pH 8.0, 50 mM NaCl, 10 mM MgCl2 , 1 mM dithiothreitol) DNase I recombinant, RNase free (10 U/μL) and incubation Buffer (10X) (Roche Diagnostics) dNTP, 10 mM (Roche Diagnostics) Dynal Streptavidin beads (Invitrogen Dynal M-280) EDTA (50 mM, pH 8.0) Ethanol (70% and 100%) Ethidium bromide(10 mg/ml) Glycogen (20 mg/mL) (Roche Diagnostics) InCert low melt agarose (1%, melted in sterile 50 mM EDTA pH 8.0 and stored at 4 °C) (Lonza) LIDS Buffer (10 mM Tris-Cl, pH 8.0,1% Lauryl sulfate lithium salt (Sigma), 100 mM EDTA) MmeI (2 U/μL, New England Biolabs) NaOAc (3 M, pH 5.3) NaOH (0.15 M) NEB Buffer 2 (10X; New England Biolabs) NEB Buffer 4 (10X; New England Biolabs) Igepal CA-630 (Sigma) Phenol (Invitrogen) Phenol: chloroform: Isoamyl Alcohol (25:24:1, Invitrogen) Phosphatase alkaline, shrimp (SAP; 1 U/μL, Roche Diagnostics) Phosphate Buffered Saline without Mg2+ /Ca2+ (1X, pH 7.2, GIBCO) Phusion DNA Polymerase (2 U/μL, Finnzymes) and Phusion HF reaction Buffer (5X) RSB Buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 ) S-adenosylmethionine (SAM; 500 μM, New England Biolabs) Spin-X filter (Fisher) T4 DNA Polymerase (New England Biolabs) TBE buffer (0.5X; 40 mM Tris-Cl, pH 8.3, 45 mM Boric Acid, 1 mM EDTA) TE Buffer (1X; 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) Tris-HCl buffer (1X, pH8.0) T4 Ligase (5 U/μL) and ligation buffer (10X) (Roche Diagnostics) Trypan blue (Gibco) Ultrapure™ Agarose (Invitrogen) Ultrapure™ L.M.P. .. Agarose (Invitrogen) Yeast Chromosome PFG molecular weight marker (New England Biolabs)

Recombinant:

Article Title: DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells
Article Snippet: .. Baker) DNA ladder (25 bp) (Invitrogen) DNA Polymerase Buffer (1X; 10 mM Tris-Cl, pH 8.0, 50 mM NaCl, 10 mM MgCl2 , 1 mM dithiothreitol) DNase I recombinant, RNase free (10 U/μL) and incubation Buffer (10X) (Roche Diagnostics) dNTP, 10 mM (Roche Diagnostics) Dynal Streptavidin beads (Invitrogen Dynal M-280) EDTA (50 mM, pH 8.0) Ethanol (70% and 100%) Ethidium bromide(10 mg/ml) Glycogen (20 mg/mL) (Roche Diagnostics) InCert low melt agarose (1%, melted in sterile 50 mM EDTA pH 8.0 and stored at 4 °C) (Lonza) LIDS Buffer (10 mM Tris-Cl, pH 8.0,1% Lauryl sulfate lithium salt (Sigma), 100 mM EDTA) MmeI (2 U/μL, New England Biolabs) NaOAc (3 M, pH 5.3) NaOH (0.15 M) NEB Buffer 2 (10X; New England Biolabs) NEB Buffer 4 (10X; New England Biolabs) Igepal CA-630 (Sigma) Phenol (Invitrogen) Phenol: chloroform: Isoamyl Alcohol (25:24:1, Invitrogen) Phosphatase alkaline, shrimp (SAP; 1 U/μL, Roche Diagnostics) Phosphate Buffered Saline without Mg2+ /Ca2+ (1X, pH 7.2, GIBCO) Phusion DNA Polymerase (2 U/μL, Finnzymes) and Phusion HF reaction Buffer (5X) RSB Buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 ) S-adenosylmethionine (SAM; 500 μM, New England Biolabs) Spin-X filter (Fisher) T4 DNA Polymerase (New England Biolabs) TBE buffer (0.5X; 40 mM Tris-Cl, pH 8.3, 45 mM Boric Acid, 1 mM EDTA) TE Buffer (1X; 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) Tris-HCl buffer (1X, pH8.0) T4 Ligase (5 U/μL) and ligation buffer (10X) (Roche Diagnostics) Trypan blue (Gibco) Ultrapure™ Agarose (Invitrogen) Ultrapure™ L.M.P. .. Agarose (Invitrogen) Yeast Chromosome PFG molecular weight marker (New England Biolabs)

Ligation:

Article Title: DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells
Article Snippet: .. Baker) DNA ladder (25 bp) (Invitrogen) DNA Polymerase Buffer (1X; 10 mM Tris-Cl, pH 8.0, 50 mM NaCl, 10 mM MgCl2 , 1 mM dithiothreitol) DNase I recombinant, RNase free (10 U/μL) and incubation Buffer (10X) (Roche Diagnostics) dNTP, 10 mM (Roche Diagnostics) Dynal Streptavidin beads (Invitrogen Dynal M-280) EDTA (50 mM, pH 8.0) Ethanol (70% and 100%) Ethidium bromide(10 mg/ml) Glycogen (20 mg/mL) (Roche Diagnostics) InCert low melt agarose (1%, melted in sterile 50 mM EDTA pH 8.0 and stored at 4 °C) (Lonza) LIDS Buffer (10 mM Tris-Cl, pH 8.0,1% Lauryl sulfate lithium salt (Sigma), 100 mM EDTA) MmeI (2 U/μL, New England Biolabs) NaOAc (3 M, pH 5.3) NaOH (0.15 M) NEB Buffer 2 (10X; New England Biolabs) NEB Buffer 4 (10X; New England Biolabs) Igepal CA-630 (Sigma) Phenol (Invitrogen) Phenol: chloroform: Isoamyl Alcohol (25:24:1, Invitrogen) Phosphatase alkaline, shrimp (SAP; 1 U/μL, Roche Diagnostics) Phosphate Buffered Saline without Mg2+ /Ca2+ (1X, pH 7.2, GIBCO) Phusion DNA Polymerase (2 U/μL, Finnzymes) and Phusion HF reaction Buffer (5X) RSB Buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 ) S-adenosylmethionine (SAM; 500 μM, New England Biolabs) Spin-X filter (Fisher) T4 DNA Polymerase (New England Biolabs) TBE buffer (0.5X; 40 mM Tris-Cl, pH 8.3, 45 mM Boric Acid, 1 mM EDTA) TE Buffer (1X; 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) Tris-HCl buffer (1X, pH8.0) T4 Ligase (5 U/μL) and ligation buffer (10X) (Roche Diagnostics) Trypan blue (Gibco) Ultrapure™ Agarose (Invitrogen) Ultrapure™ L.M.P. .. Agarose (Invitrogen) Yeast Chromosome PFG molecular weight marker (New England Biolabs)

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  • 92
    Roche pcr buffer
    Methylation status of MGMT promoter gene <t>(MS-PCR)</t> in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control <t>DNA,</t> PC positive control DNA, MW molecular weight marks (100-bp ladder)
    Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr buffer/product/Roche
    Average 92 stars, based on 56 article reviews
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    pcr buffer - by Bioz Stars, 2020-05
    92/100 stars
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    97
    Roche dnase i
    <t>DNase</t> I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His 6 concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His 6 . A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His 6 is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.
    Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 2243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Roche
    Average 97 stars, based on 2243 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-05
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    Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Journal: Clinical Epigenetics

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

    doi: 10.1186/s13148-018-0594-9

    Figure Lengend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Article Snippet: PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

    Techniques: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

    Dnmt1 Inhibits the Expression of Recombinant GFP Genes Wild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression. (A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods. (B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP + cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells (C) The histogram showing the fraction of GFP + cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP + cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p

    Journal: PLoS Genetics

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    doi: 10.1371/journal.pgen.0030110

    Figure Lengend Snippet: Dnmt1 Inhibits the Expression of Recombinant GFP Genes Wild-type or Dnmt1−/− ES cells carrying DR-GFP were transfected with the I-SceI expression vector and PSVbGal, grown 4 d, and analyzed for GFP recombination and expression. (A) Genomic DNA from the two cell lines was PCR-amplified with nonrecombinant (5′-unrec) and recombinant (5′-rec) primers. The specificity of the products and the linearity of the reactions were controlled as described in Materials and Methods. qPCR of the same samples was carried out as described in Materials and Methods. (B) FACS analysis of cells transfected with I-SceI is shown. The gating of GFP + cells was created to exclude up the 99.5% of wild-type untransfected ES cells. The same gating applied to Dnmt1−/− cells shows a significant increase in the population expressing GFP. Following I-SceI transfection, Dnmt1−/− cells were treated with 5-AzadC as described in Materials and Methods. Treatment with 5-AzadC increased the fraction of cells expressing GFP in wild-type ES but did not enhance the expression of GFP in the Dnmt1−/− cells (C) The histogram showing the fraction of GFP + cells derived from three experiments is shown. To obtain reliable values of differential GFP fluorescence in ES and Dnmt1−/− cells, we compared the percentage of GFP + cells, normalized for the transfection efficiency in six experiments (three in duplicate), with the Wilcoxon Kruskal-Wallis Test, *, p

    Article Snippet: PCR was performed in a 50-μl reaction mixture containing 2 μl of synthesized cDNA product or 0.5 μg of genomic DNA, 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.3 mM dNTP, 1.25 unit of Taq polymerase (Roche, http://www.roche.com ), and 6 μM of each primer.

    Techniques: Expressing, Recombinant, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, FACS, Derivative Assay, Fluorescence

    Dnmt1 Selectively Binds Recombinant GFP Chromatin Hela cells carrying DR-GFP were transfected with I-SceI and treated 24 h later with 1 μM 5-AzadC for 1, 2, and 4 d. Chromatin immunoprecipitation (ChIp) was carried out as described in Materials and Methods. (A) PCR of immunoprecipitated DNA with antibodies to Dnmt1 is shown. None indicates chromatin derived from cells transfected with control plasmid, (−) or (+) indicates the treatment with 5-AzadC. Rec, unrec indicate the primers used for amplification. The lower panel shows the statistical analysis of PCR reactions carried out at 25 and 30 cycles when the reactions with the three sets of primers were in the linear range. Immunoprecipitations were carried out with nonspecific immunoglobulin G (Control immunoglobulin G) and anti-Dnmt1 specific antibodies. The primers used were: (1) unrec; (2) rec; and (3) actin (*, p

    Journal: PLoS Genetics

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    doi: 10.1371/journal.pgen.0030110

    Figure Lengend Snippet: Dnmt1 Selectively Binds Recombinant GFP Chromatin Hela cells carrying DR-GFP were transfected with I-SceI and treated 24 h later with 1 μM 5-AzadC for 1, 2, and 4 d. Chromatin immunoprecipitation (ChIp) was carried out as described in Materials and Methods. (A) PCR of immunoprecipitated DNA with antibodies to Dnmt1 is shown. None indicates chromatin derived from cells transfected with control plasmid, (−) or (+) indicates the treatment with 5-AzadC. Rec, unrec indicate the primers used for amplification. The lower panel shows the statistical analysis of PCR reactions carried out at 25 and 30 cycles when the reactions with the three sets of primers were in the linear range. Immunoprecipitations were carried out with nonspecific immunoglobulin G (Control immunoglobulin G) and anti-Dnmt1 specific antibodies. The primers used were: (1) unrec; (2) rec; and (3) actin (*, p

    Article Snippet: PCR was performed in a 50-μl reaction mixture containing 2 μl of synthesized cDNA product or 0.5 μg of genomic DNA, 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.3 mM dNTP, 1.25 unit of Taq polymerase (Roche, http://www.roche.com ), and 6 μM of each primer.

    Techniques: Recombinant, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Derivative Assay, Plasmid Preparation, Amplification

    DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p

    Journal: PLoS Genetics

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    doi: 10.1371/journal.pgen.0030110

    Figure Lengend Snippet: DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p

    Article Snippet: PCR was performed in a 50-μl reaction mixture containing 2 μl of synthesized cDNA product or 0.5 μg of genomic DNA, 5 μl of 10× PCR buffer, 1.5 mM MgCl2, 0.3 mM dNTP, 1.25 unit of Taq polymerase (Roche, http://www.roche.com ), and 6 μM of each primer.

    Techniques: DNA Methylation Assay, Recombination Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, FACS, Inhibition, Methylation, Fluorescence

    Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Journal: Clinical Epigenetics

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

    doi: 10.1186/s13148-018-0594-9

    Figure Lengend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Article Snippet: PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

    Techniques: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

    DNase I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His 6 concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His 6 . A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His 6 is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.

    Journal: Nucleic Acids Research

    Article Title: Toxin-antitoxin regulation: bimodal interaction of YefM-YoeB with paired DNA palindromes exerts transcriptional autorepression

    doi: 10.1093/nar/gkl1028

    Figure Lengend Snippet: DNase I footprinting of the yefM-yoeB promoter-operator region. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either upper or lower strands. YefM concentrations (μM, left to right): 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0. YefM–YoeB–His 6 concentrations (μM, left to right): 0, 0.007, 0.018, 0.036, 0.072, 0.18, 0.36, 0.72, 1.8 and 3.6. The locations of the L and S repeats are marked by inverted arrows. Shaded boxes denote the regions protected from DNase I digestion by YefM and YefM–YoeB–His 6 . A + G, Maxam–Gilbert sequencing reactions. A position on the lower strand that is hypersenstive to DNase I cleavage in the presence of YefM and YefM–YoeB–His 6 is highlighted by the star. The relative dispositions of regions on the upper and lower strands that are protected from DNase I digestion and other features of the yefM-yoeB promoter–operator region are illustrated in the lower panel.

    Article Snippet: Each reaction was treated with 0.0075 U of DNase I (Roche, RNase free, 10 U/μl diluted in buffer [20 mM Tris–HCl (pH 7.5), 50 mM NaCl, 7.5 mM MgCl2 and 5 mM CaCl2 ]) for 45 s at 22°C.

    Techniques: Footprinting, Polymerase Chain Reaction, Sequencing