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  • 95
    Qiagen minelute pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, <t>MinElute</t> PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Minelute Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 16148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher genejet pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, <t>MinElute</t> PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Genejet Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Qiagen polymerase chain reaction purification kit
    APC is a direct HIF-1α target. (A) ChIP analysis for HIF-1α binding to the APC and CA9 promoters. U2OS cells were exposed to 1% O 2 for the indicated periods before fixation and lysis. HIF-1α-bound <t>DNA</t> was amplified with specific primers spanning the annotated HRE region on the CA9 and the putative HRE at the APC promoter. Rabbit immunoglobulin G (IgG) was used as a control for the immunoprecipitation. Input represents 10% of the starting material used per immunoprecipitation (IP). (B) ChIP analysis for HIF-1β binding at the APC and CA9 promoters. U2OS cells were exposed to 1% O 2 for 24 h before fixation and lysis. HIF-1β–bound DNA was amplified with specific primers spanning the annotated HRE region on the CA9 and the putative HRE at the APC promoter. Rabbit IgG was used as a control for the immunoprecipitation. Input represents 10% of the starting material used per IP. (C) ChIP for AcH3. (D) Polymerase II at the APC HRE site. Cells were treated and processed as described in B. Rabbit IgG was used as a negative control. APC HRE site was amplified using specific <t>PCR</t> primers. Inputs represent 10% of starting material used in each IP.
    Polymerase Chain Reaction Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 81/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Qiagen polymerase chain reaction clean up kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Polymerase Chain Reaction Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 79/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Qiagen minielute polymerase chain reaction pcr purification kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Minielute Polymerase Chain Reaction Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 87/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Qiagen qiaquick1 polymerase chain reaction pcr extraction kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Qiaquick1 Polymerase Chain Reaction Pcr Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen minelute 96 uf pcr purification plates
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Minelute 96 Uf Pcr Purification Plates, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher qiagen purification kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Qiagen Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen mini plasmid kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Mini Plasmid Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen plasmid midi kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Plasmid Midi Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 7316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen large construct dna purification kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Large Construct Dna Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen plasmid maxi plasmid dna purification kit
    Real-time PCR analysis of viral <t>DNA</t> replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout <t>bacmid</t> or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.
    Plasmid Maxi Plasmid Dna Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Qiagen total rna purification all prep dna rna mini kit qiagen
    Real-time PCR analysis of viral <t>DNA</t> replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout <t>bacmid</t> or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.
    Total Rna Purification All Prep Dna Rna Mini Kit Qiagen, supplied by Qiagen, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen pyromark pcr kit
    Real-time PCR analysis of viral <t>DNA</t> replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout <t>bacmid</t> or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.
    Pyromark Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 2816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    MACHEREY NAGEL pcr cleanup kit
    Real-time PCR analysis of viral <t>DNA</t> replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout <t>bacmid</t> or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.
    Pcr Cleanup Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 99/100, based on 646 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen pcr cloning plus kit
    Clustering of C. difficile <t>PCR-ribotypes.</t> (A) Clustering of PRC-ribotypes based on fingerprinting profiles generated by capillary gel electrophoresis-based PCR-ribotyping. Dendrogram is color coded according to MLST type. The exact lengths of the bands, representing the 16S-23S <t>rRNA</t> intergenic spacer regions are given in Table S1 . (B) Minimum spanning tree of MLST results showing relatedness of PCR-ribotypes. Each circle represents one sequence type (ST) and is subdivided into sectors corresponding to the number of PCR-ribotypes represented with this ST. The numbers between circles represent number of differing loci between the STs.
    Pcr Cloning Plus Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen pcr cloningplus kit
    Clustering of C. difficile <t>PCR-ribotypes.</t> (A) Clustering of PRC-ribotypes based on fingerprinting profiles generated by capillary gel electrophoresis-based PCR-ribotyping. Dendrogram is color coded according to MLST type. The exact lengths of the bands, representing the 16S-23S <t>rRNA</t> intergenic spacer regions are given in Table S1 . (B) Minimum spanning tree of MLST results showing relatedness of PCR-ribotypes. Each circle represents one sequence type (ST) and is subdivided into sectors corresponding to the number of PCR-ribotypes represented with this ST. The numbers between circles represent number of differing loci between the STs.
    Pcr Cloningplus Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen long range pcr kit
    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
    Long Range Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen type it hrm pcr kit
    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
    Type It Hrm Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen quantitect probe pcr kit
    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
    Quantitect Probe Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen miscript pcr starter kit
    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
    Miscript Pcr Starter Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the <t>PCR</t> products that were amplified from the IL28A and IL28B promoters that included the <t>SNP</t> rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .
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    ChIP-chip and ChIP-qPCR analysis of FoxP3 bound DNA from CD4 + CD25 hi human T reg cells. Anti-FoxP3 or control rabbit Ig was used to precipitate cross-linked protein–DNA complexes from expanded CD4 + CD25 hi human T reg cells lysate. The cross-linking of the immunoprecipitated material was removed and protease-treated and the DNA was purified and amplified. The resultant material was hybridized to the whole genome using GeneChip Human tiling 1.0R array set to identify the locations of binding sites for FoxP3. Two sets of graphs: FoxP3 IP versus the Ig control and FoxP3 IP versus Input DNA were generated on the hs.NCBIv35 version of the genome essentially following the method described in Cawley et el. (reference 50 ). (a) Signal enrichment graphs of IL-7R locus (chr5:35863179-35918811). Several regions in IL-7R locus are predicted to be positive (chr5:35892564-35892809 promoter) and negative (chr5:35890618-35890846 2K upstream; chr5:35907667-35907852 Intron 4; chr5:35911721-35911888 intron 7 and exon 8). (b) <t>SYBR</t> green qPCR of IL-7R chromosomal regions. FoxP3 IP versus the IgG fold enrichment ratio was determined from duplicate ChIP assay evaluated in duplicate by real time <t>PCR.</t>
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    HIF1α is a key transcription factor upregulating HK2 expression in EML4-ALK-expressing lung cancer cells ( A ) The HK2 luciferase reporter pGL2-138HK2-Luc or the HRE mutant form was co-transfected into A549 cells along with pCDNA3.1/Zeo(+) (vector) or pCDNA3.1/Zeo(+)-EML4-ALK. The transactivation of the HK2 promoter by co-transfected ALK was measured by luciferase assays. The luciferase activities were presented as fold changes relative to the value of the vector control cells (defined as 1 fold). ChIP assay was performed to assess the binding of HIF1α to the HK2 promoter in ALK knockdown and control cells of H2228 and H3122 and in A549 cells transduced to express EML4-ALK. The HK2 promoter DNA sequence co-precipitated with HIF1α was analyzed by qPCR using <t>SYBR</t> Green ( B ) and regular <t>PCR</t> ( C ) using the primers listed in Table 1 . Fractions of immunoprecipitates and total cellular DNA were also analyzed with regular PCR amplification of the HK2 promoter..
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    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin.

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    APC is a direct HIF-1α target. (A) ChIP analysis for HIF-1α binding to the APC and CA9 promoters. U2OS cells were exposed to 1% O 2 for the indicated periods before fixation and lysis. HIF-1α-bound DNA was amplified with specific primers spanning the annotated HRE region on the CA9 and the putative HRE at the APC promoter. Rabbit immunoglobulin G (IgG) was used as a control for the immunoprecipitation. Input represents 10% of the starting material used per immunoprecipitation (IP). (B) ChIP analysis for HIF-1β binding at the APC and CA9 promoters. U2OS cells were exposed to 1% O 2 for 24 h before fixation and lysis. HIF-1β–bound DNA was amplified with specific primers spanning the annotated HRE region on the CA9 and the putative HRE at the APC promoter. Rabbit IgG was used as a control for the immunoprecipitation. Input represents 10% of the starting material used per IP. (C) ChIP for AcH3. (D) Polymerase II at the APC HRE site. Cells were treated and processed as described in B. Rabbit IgG was used as a negative control. APC HRE site was amplified using specific PCR primers. Inputs represent 10% of starting material used in each IP.

    Journal: Molecular Biology of the Cell

    Article Title: Adenomatous Polyposis Coli and Hypoxia-inducible Factor-1? Have an Antagonistic Connection

    doi: 10.1091/mbc.E10-04-0312

    Figure Lengend Snippet: APC is a direct HIF-1α target. (A) ChIP analysis for HIF-1α binding to the APC and CA9 promoters. U2OS cells were exposed to 1% O 2 for the indicated periods before fixation and lysis. HIF-1α-bound DNA was amplified with specific primers spanning the annotated HRE region on the CA9 and the putative HRE at the APC promoter. Rabbit immunoglobulin G (IgG) was used as a control for the immunoprecipitation. Input represents 10% of the starting material used per immunoprecipitation (IP). (B) ChIP analysis for HIF-1β binding at the APC and CA9 promoters. U2OS cells were exposed to 1% O 2 for 24 h before fixation and lysis. HIF-1β–bound DNA was amplified with specific primers spanning the annotated HRE region on the CA9 and the putative HRE at the APC promoter. Rabbit IgG was used as a control for the immunoprecipitation. Input represents 10% of the starting material used per IP. (C) ChIP for AcH3. (D) Polymerase II at the APC HRE site. Cells were treated and processed as described in B. Rabbit IgG was used as a negative control. APC HRE site was amplified using specific PCR primers. Inputs represent 10% of starting material used in each IP.

    Article Snippet: Supernatant was incubated with specific antibodies overnight and then with protein G-Sepharose beads for 1 h. After extensive wash step, the complexes were eluted with buffer (100 mmol/l NaHCO3 and 1% SDS) and incubated with proteinase K. DNA was purified using polymerase chain reaction (PCR) purification kit (QIAGEN/National Blood Service, Burmingham, United Kingdom).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Lysis, Amplification, Immunoprecipitation, Negative Control, Polymerase Chain Reaction

    Analysis of DNA extraction efficacy using regenerated columns from gel extraction kits. ( A ) qPCR analysis for the residual DNA from Qiagen columns regenerated using different reagents. LIF-contaminated columns were cleaned with the indicated reagents based on the protocols in Fig. 1A . Thirty microlitres of eluate was obtained from the regenerated columns, 1 μL of which was used for qPCR. The data were processed and presented as in Fig. 1C . ( B ) Comparison of the DNA extraction efficacy between the regenerated and fresh columns from gel extraction kits. A 50 μL PCR reaction was used for agarose gel electrophoresis, and DNA extraction was performed using the regenerated or fresh columns according to the gel extraction kit manual. The extracted DNA was quantified with a Nanophotometer and verified by agarose gel electrophoresis as shown in Fig. 2C . The full-length image is presented in Supplementary Fig. S3 . ( C ) The regenerated columns showed a comparable ability to purify different sizes of DNA as the fresh columns. A 50 μL PCR reaction was used for agarose gel electrophoresis, which was purified and measured as in ( B ). ( D ) Comparison of DNA purification efficacy between the regenerated and fresh columns from different commercial kits. A 50 μL LIF PCR reaction was used for agarose gel electrophoresis, and the DNA was extracted from the gel with regenerated or fresh columns from different gel extraction kits. The data were processed and presented as in B. Each column in A and C represents the mean ± SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Rapid Regeneration and Reuse of Silica Columns from PCR Purification and Gel Extraction Kits

    doi: 10.1038/s41598-018-30316-w

    Figure Lengend Snippet: Analysis of DNA extraction efficacy using regenerated columns from gel extraction kits. ( A ) qPCR analysis for the residual DNA from Qiagen columns regenerated using different reagents. LIF-contaminated columns were cleaned with the indicated reagents based on the protocols in Fig. 1A . Thirty microlitres of eluate was obtained from the regenerated columns, 1 μL of which was used for qPCR. The data were processed and presented as in Fig. 1C . ( B ) Comparison of the DNA extraction efficacy between the regenerated and fresh columns from gel extraction kits. A 50 μL PCR reaction was used for agarose gel electrophoresis, and DNA extraction was performed using the regenerated or fresh columns according to the gel extraction kit manual. The extracted DNA was quantified with a Nanophotometer and verified by agarose gel electrophoresis as shown in Fig. 2C . The full-length image is presented in Supplementary Fig. S3 . ( C ) The regenerated columns showed a comparable ability to purify different sizes of DNA as the fresh columns. A 50 μL PCR reaction was used for agarose gel electrophoresis, which was purified and measured as in ( B ). ( D ) Comparison of DNA purification efficacy between the regenerated and fresh columns from different commercial kits. A 50 μL LIF PCR reaction was used for agarose gel electrophoresis, and the DNA was extracted from the gel with regenerated or fresh columns from different gel extraction kits. The data were processed and presented as in B. Each column in A and C represents the mean ± SD of three independent experiments. * p

    Article Snippet: Establishment of a regeneration method for disposable silica columns from a PCR purification kit To establish the method for regenerating used columns, fresh columns from a PCR purification kit (Qiagen) were initially used to purify a human LIF gene from PCR product.

    Techniques: DNA Extraction, Gel Extraction, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, DNA Purification

    Disposable columns from Qiagen PCR purification and gel extraction kits can be repeatedly regenerated and reused a minimum of five times. ( A ) DNA purification efficacy for the repeatedly regenerated PCR purification kit columns. DNA-contaminated columns were cleaned with 1 M phosphoric acid and used for PCR product (TBox 5) purification; the same columns were regenerated and reused four additional times. The concentration of obtained DNA for each cycle was measured with a Nanophotometer and the data are presented as gel pictures (left) and in a graph (right). ( B ) The analysis of DNA extraction efficacy for the repeatedly regenerated gel extraction kit columns. DNA-contaminated columns were regenerated as in A, where a 50 μL PCR reaction was used for agarose gel electrophoresis. DNA extraction from agarose gel was performed according to the gel extraction kit manual. The data were processed as in A. The full length images for A (left) and B (left) are presented in Supplementary Fig. S4A,B . ( C ) Comparison of DNA purification efficacy between lab-made and commercial buffers. The lab-made buffers (LB) were used for DNA purification for regenerated Qiagen columns, whereas the commercial buffers (CB) were used for DNA purification for the fresh Qiagen or Axygen kit columns. The assays were performed as shown in Fig. 2C . ( D ) Comparison of DNA extraction efficacy between lab-made and commercial buffers. The lab-made buffers (LB) were used for DNA extraction from agarose gel using regenerated Qiagen columns, whereas the commercial buffers (CB) were used for DNA extraction from agarose gel using fresh Qiagen or Axygen kit columns. The assays were performed as shown in Fig. 3B . The protocol for DNA purification with lab-made buffers is described in the materials and methods section. Each column in A (right) and B (right) represents the mean ± SD of three independent experiments.

    Journal: Scientific Reports

    Article Title: Rapid Regeneration and Reuse of Silica Columns from PCR Purification and Gel Extraction Kits

    doi: 10.1038/s41598-018-30316-w

    Figure Lengend Snippet: Disposable columns from Qiagen PCR purification and gel extraction kits can be repeatedly regenerated and reused a minimum of five times. ( A ) DNA purification efficacy for the repeatedly regenerated PCR purification kit columns. DNA-contaminated columns were cleaned with 1 M phosphoric acid and used for PCR product (TBox 5) purification; the same columns were regenerated and reused four additional times. The concentration of obtained DNA for each cycle was measured with a Nanophotometer and the data are presented as gel pictures (left) and in a graph (right). ( B ) The analysis of DNA extraction efficacy for the repeatedly regenerated gel extraction kit columns. DNA-contaminated columns were regenerated as in A, where a 50 μL PCR reaction was used for agarose gel electrophoresis. DNA extraction from agarose gel was performed according to the gel extraction kit manual. The data were processed as in A. The full length images for A (left) and B (left) are presented in Supplementary Fig. S4A,B . ( C ) Comparison of DNA purification efficacy between lab-made and commercial buffers. The lab-made buffers (LB) were used for DNA purification for regenerated Qiagen columns, whereas the commercial buffers (CB) were used for DNA purification for the fresh Qiagen or Axygen kit columns. The assays were performed as shown in Fig. 2C . ( D ) Comparison of DNA extraction efficacy between lab-made and commercial buffers. The lab-made buffers (LB) were used for DNA extraction from agarose gel using regenerated Qiagen columns, whereas the commercial buffers (CB) were used for DNA extraction from agarose gel using fresh Qiagen or Axygen kit columns. The assays were performed as shown in Fig. 3B . The protocol for DNA purification with lab-made buffers is described in the materials and methods section. Each column in A (right) and B (right) represents the mean ± SD of three independent experiments.

    Article Snippet: Establishment of a regeneration method for disposable silica columns from a PCR purification kit To establish the method for regenerating used columns, fresh columns from a PCR purification kit (Qiagen) were initially used to purify a human LIF gene from PCR product.

    Techniques: Polymerase Chain Reaction, Purification, Gel Extraction, DNA Purification, Concentration Assay, DNA Extraction, Agarose Gel Electrophoresis

    Phosphoric acid is the most effective reagent for eliminating residual DNA from used Qiagen PCR purification kit columns. ( A ) A scheme showing the workflow for different protocols used in this study. ( B ) The standard curve that was plotted with the Ct values against –Log 10 DNA concentration (ng/μL). Ten microliters of LIF DNA template at a concentration of 100 ng/μL was consecutively diluted at 1/10 for 8 times, and the diluted samples (starting from 1 * 10 −2 ng/μL) were used for qPCR. Ct: Threshold cycles in quantitative PCR. ( C ) qPCR analysis to assess the amount of residual DNA eluted from the columns that were cleaned with different chemical reagents. LIF-contaminated columns were cleaned with indicated reagent based on protocol I. One microlitre of eluate was used to assess the presence of residual DNA by qPCR, with each sample assayed in triplicate. The obtained Ct values were converted into DNA concentration using the standard curve in B and were subjected to statistical analysis. The value of each column within the graph is presented in the bottom of the panel to clearly show the differences between the samples. ( D ) qPCR results for the amount of residual DNA from the columns that were cleaned with DNase I or acidic phenol. Contaminated columns were cleaned based on protocol II or III. qPCR assays were performed as in C. (E) qPCR showing DNA elimination for the used columns cleaned with different concentrations of phosphoric acid. Contaminated columns were cleaned with different concentrations of phosphoric acid as detailed in protocol I. qPCR assays performed as in C. Each column in C–E represents the mean ± SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Rapid Regeneration and Reuse of Silica Columns from PCR Purification and Gel Extraction Kits

    doi: 10.1038/s41598-018-30316-w

    Figure Lengend Snippet: Phosphoric acid is the most effective reagent for eliminating residual DNA from used Qiagen PCR purification kit columns. ( A ) A scheme showing the workflow for different protocols used in this study. ( B ) The standard curve that was plotted with the Ct values against –Log 10 DNA concentration (ng/μL). Ten microliters of LIF DNA template at a concentration of 100 ng/μL was consecutively diluted at 1/10 for 8 times, and the diluted samples (starting from 1 * 10 −2 ng/μL) were used for qPCR. Ct: Threshold cycles in quantitative PCR. ( C ) qPCR analysis to assess the amount of residual DNA eluted from the columns that were cleaned with different chemical reagents. LIF-contaminated columns were cleaned with indicated reagent based on protocol I. One microlitre of eluate was used to assess the presence of residual DNA by qPCR, with each sample assayed in triplicate. The obtained Ct values were converted into DNA concentration using the standard curve in B and were subjected to statistical analysis. The value of each column within the graph is presented in the bottom of the panel to clearly show the differences between the samples. ( D ) qPCR results for the amount of residual DNA from the columns that were cleaned with DNase I or acidic phenol. Contaminated columns were cleaned based on protocol II or III. qPCR assays were performed as in C. (E) qPCR showing DNA elimination for the used columns cleaned with different concentrations of phosphoric acid. Contaminated columns were cleaned with different concentrations of phosphoric acid as detailed in protocol I. qPCR assays performed as in C. Each column in C–E represents the mean ± SD of three independent experiments. * p

    Article Snippet: Establishment of a regeneration method for disposable silica columns from a PCR purification kit To establish the method for regenerating used columns, fresh columns from a PCR purification kit (Qiagen) were initially used to purify a human LIF gene from PCR product.

    Techniques: Polymerase Chain Reaction, Purification, Concentration Assay, Real-time Polymerase Chain Reaction

    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Journal: Epigenetics & Chromatin

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination

    doi: 10.1186/s13072-018-0247-4

    Figure Lengend Snippet: TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Article Snippet: Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick).

    Techniques: Cell Culture, Purification, Immunoprecipitation, Polymerase Chain Reaction

    Seizures in vitro increase binding of FKHR to the bim promoter, and Bim antisense is neuroprotective. (A) Representative Western blots from hippocampal neuronal cultures at 24 hours, showing that seizurelike activity in vitro causes FKHR/FKHRL-1 dephosphorylation and increased Bim expression. Immunoblots showing unaffected Bid and α-tubulin levels are included as controls. Immunoblots are representative of two or three independent experiments. (B) Chromatin immunoprecipitation assay demonstrating immunoprecipitation of the bim promoter (pmr) by FKHR. Chromatin fragments were immunoprecipitated using either anti-FKHR or anti–phospho-FKHR followed by PCR with primers specific for the bim -FKHR promoter region. Genomic DNA was subject to PCR as a positive (+) control. Phospho-FKHR did not precipitate the bim promoter. In contrast, FKHR precipitated the bim promoter, and this was increased in cultures subject to seizures and 1 hour of recovery. The image is representative of two independent experiments. (C) Representative Western blots confirming that Bim antisense (AS) reduces Bim levels compared with those in control cultures treated with a scrambled (scram) sequence. Immunoblots showing unaffected Bid and α-tubulin levels are included as controls. Immunoblots are representative of two independent experiments. (D) Graph showing effects of Bim antisense on seizure-induced LDH release. Injury assessment at 24 hours revealed that seizures (seiz) elevated LDH levels to about 275% of control (base-line) levels. Incubation of cultures with the scrambled Bim antisense did not alter seizure-induced LDH release. In contrast, Bim antisense significantly reduced LDH release following seizures. * P

    Journal: Journal of Clinical Investigation

    Article Title: Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy

    doi: 10.1172/JCI200419971

    Figure Lengend Snippet: Seizures in vitro increase binding of FKHR to the bim promoter, and Bim antisense is neuroprotective. (A) Representative Western blots from hippocampal neuronal cultures at 24 hours, showing that seizurelike activity in vitro causes FKHR/FKHRL-1 dephosphorylation and increased Bim expression. Immunoblots showing unaffected Bid and α-tubulin levels are included as controls. Immunoblots are representative of two or three independent experiments. (B) Chromatin immunoprecipitation assay demonstrating immunoprecipitation of the bim promoter (pmr) by FKHR. Chromatin fragments were immunoprecipitated using either anti-FKHR or anti–phospho-FKHR followed by PCR with primers specific for the bim -FKHR promoter region. Genomic DNA was subject to PCR as a positive (+) control. Phospho-FKHR did not precipitate the bim promoter. In contrast, FKHR precipitated the bim promoter, and this was increased in cultures subject to seizures and 1 hour of recovery. The image is representative of two independent experiments. (C) Representative Western blots confirming that Bim antisense (AS) reduces Bim levels compared with those in control cultures treated with a scrambled (scram) sequence. Immunoblots showing unaffected Bid and α-tubulin levels are included as controls. Immunoblots are representative of two independent experiments. (D) Graph showing effects of Bim antisense on seizure-induced LDH release. Injury assessment at 24 hours revealed that seizures (seiz) elevated LDH levels to about 275% of control (base-line) levels. Incubation of cultures with the scrambled Bim antisense did not alter seizure-induced LDH release. In contrast, Bim antisense significantly reduced LDH release following seizures. * P

    Article Snippet: DNA samples were purified using a PCR clean-up kit (QIAGEN Inc., Valencia, California, USA), and then DNA was subjected to 36 cycles of PCR with primers specific to the FKHR consensus on the bim promoter: sense 5′-TCTCTCAACTGTCCTTCC-3′, antisense 5′-TGTTTACCCTAGTGACTTGG-3′ (accession number ) ( ).

    Techniques: In Vitro, Binding Assay, Western Blot, Activity Assay, De-Phosphorylation Assay, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Sequencing, Incubation

    Identification of uspA genes from M. catarrhalis CEACAM-binding variants. PCR of Mx D1, D2, 035E and MX2 for uspa1 (upper panel) and uspA2 (lower panel). Compared to 035E, uspA1 showed an increase in size in D1, whilst uspA2 of D2 was larger than the parental 035E. Note uspA1 of D1was larger than both 035E and MX2 genes. Data are representative of PCR products obtained on several occasions.

    Journal: PLoS ONE

    Article Title: A Novel Group of Moraxella catarrhalis UspA Proteins Mediates Cellular Adhesion via CEACAMs and Vitronectin

    doi: 10.1371/journal.pone.0045452

    Figure Lengend Snippet: Identification of uspA genes from M. catarrhalis CEACAM-binding variants. PCR of Mx D1, D2, 035E and MX2 for uspa1 (upper panel) and uspA2 (lower panel). Compared to 035E, uspA1 showed an increase in size in D1, whilst uspA2 of D2 was larger than the parental 035E. Note uspA1 of D1was larger than both 035E and MX2 genes. Data are representative of PCR products obtained on several occasions.

    Article Snippet: Genes were amplified using uspa1 and uspa2 primer pairs ( ) purified using a PCR cleanup kit (Qiagen).

    Techniques: Binding Assay, Polymerase Chain Reaction

    Identification of uspA genes from Mx CEACAM-binding variants. PCR of Mx S11N:1, S1:4, S36:1, S43:4, S45:5 and MX1 as indicated for uspA1 (upper panel) and uspA2 (lower panel). uspA1 PCR gave no bands for uspA1 in strains S1:4, S36:1 and S43:4 whereas larger than expected bands were observed for S11N:1, S45:5 and MX1. PCR products were obtained for uspA2 for all strains tested. Data are representative of PCR products obtained on several occasions.

    Journal: PLoS ONE

    Article Title: A Novel Group of Moraxella catarrhalis UspA Proteins Mediates Cellular Adhesion via CEACAMs and Vitronectin

    doi: 10.1371/journal.pone.0045452

    Figure Lengend Snippet: Identification of uspA genes from Mx CEACAM-binding variants. PCR of Mx S11N:1, S1:4, S36:1, S43:4, S45:5 and MX1 as indicated for uspA1 (upper panel) and uspA2 (lower panel). uspA1 PCR gave no bands for uspA1 in strains S1:4, S36:1 and S43:4 whereas larger than expected bands were observed for S11N:1, S45:5 and MX1. PCR products were obtained for uspA2 for all strains tested. Data are representative of PCR products obtained on several occasions.

    Article Snippet: Genes were amplified using uspa1 and uspa2 primer pairs ( ) purified using a PCR cleanup kit (Qiagen).

    Techniques: Binding Assay, Polymerase Chain Reaction

    A schematic representation of the IVC screen protocol. The lower section of the diagram shows the molecules bound to the surface of the streptavidin-coated beads at each step. Blue rectangles represent template DNA, green split arrows represent biotinylated anti-HA antibodies, turquoise clouds represent 3xHA-tagged hydrogenase proteins, blue and magenta circles represent C 12 -resazurin and C 12 -resorufin. A. DNA-bound beads are incubated with biotinylated anti-HA antibodies, added to a cell-free protein synthesis mixture, and emulsified into an oil phase. Emulsion CFPS transcribes mRNA from the bound DNA templates and synthesizes HA-tagged hydrogenase proteins, which are bound by the antibodies. The beads are then recovered from the emulsion, washed, and exposed to oxygen to challenge the hydrogenase mutants. The beads are then re-emulsified, and C 12 -resazurin is delivered to the emulsion droplets. Mutant hydrogenases that survive oxygen exposure consume hydrogen and reduce C 12 -resazurin to fluorescent C 12 -resorufin, which adsorbs to the bead surface. The beads are recovered and sorted by FACS. Fluorescent beads are added to a PCR mixture and thermocycled to recover DNA encoding improved hydrogenase mutants. B. An emulsion PCR step amplifies unique mutant templates to amounts sufficient to result in strong fluorescent signals. Beads are first incubated with biotinylated primers (represented by short black lines) and less than one molecule of template DNA per bead, then added to a PCR mixture and emulsified. The emulsion is thermocycled, and the beads are recovered, washed, and screened with the procedure shown in A .

    Journal: PLoS ONE

    Article Title: Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

    doi: 10.1371/journal.pone.0015275

    Figure Lengend Snippet: A schematic representation of the IVC screen protocol. The lower section of the diagram shows the molecules bound to the surface of the streptavidin-coated beads at each step. Blue rectangles represent template DNA, green split arrows represent biotinylated anti-HA antibodies, turquoise clouds represent 3xHA-tagged hydrogenase proteins, blue and magenta circles represent C 12 -resazurin and C 12 -resorufin. A. DNA-bound beads are incubated with biotinylated anti-HA antibodies, added to a cell-free protein synthesis mixture, and emulsified into an oil phase. Emulsion CFPS transcribes mRNA from the bound DNA templates and synthesizes HA-tagged hydrogenase proteins, which are bound by the antibodies. The beads are then recovered from the emulsion, washed, and exposed to oxygen to challenge the hydrogenase mutants. The beads are then re-emulsified, and C 12 -resazurin is delivered to the emulsion droplets. Mutant hydrogenases that survive oxygen exposure consume hydrogen and reduce C 12 -resazurin to fluorescent C 12 -resorufin, which adsorbs to the bead surface. The beads are recovered and sorted by FACS. Fluorescent beads are added to a PCR mixture and thermocycled to recover DNA encoding improved hydrogenase mutants. B. An emulsion PCR step amplifies unique mutant templates to amounts sufficient to result in strong fluorescent signals. Beads are first incubated with biotinylated primers (represented by short black lines) and less than one molecule of template DNA per bead, then added to a PCR mixture and emulsified. The emulsion is thermocycled, and the beads are recovered, washed, and screened with the procedure shown in A .

    Article Snippet: Following purification with PCR cleanup kits (Qiagen, Valencia, CA), DNA concentrations were measured with the Quant-It dsDNA broad-range assay on a Qubit fluorimeter (Invitrogen, Carlsbad, CA) following the manufacturer's protocol.

    Techniques: Incubation, Mutagenesis, FACS, Polymerase Chain Reaction

    Enrichment of beads bound to single molecules of hydrogenase DNA. Beads displaying CAT DNA and beads displaying CpI DNA were originally mixed in a 20∶1 ratio. Comparison of the PCR amplification products from nonfluorescent (−) and fluorescent (+) sorted beads by agarose gel electrophoresis indicates that the more fluorescent population is enriched in CpI-bound beads. Nonspecific amplification products are visible in both lanes.

    Journal: PLoS ONE

    Article Title: Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

    doi: 10.1371/journal.pone.0015275

    Figure Lengend Snippet: Enrichment of beads bound to single molecules of hydrogenase DNA. Beads displaying CAT DNA and beads displaying CpI DNA were originally mixed in a 20∶1 ratio. Comparison of the PCR amplification products from nonfluorescent (−) and fluorescent (+) sorted beads by agarose gel electrophoresis indicates that the more fluorescent population is enriched in CpI-bound beads. Nonspecific amplification products are visible in both lanes.

    Article Snippet: Following purification with PCR cleanup kits (Qiagen, Valencia, CA), DNA concentrations were measured with the Quant-It dsDNA broad-range assay on a Qubit fluorimeter (Invitrogen, Carlsbad, CA) following the manufacturer's protocol.

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Transfection, Staining, Confocal Microscopy

    Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Mutagenesis, Transduction, Staining, Confocal Microscopy

    Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Construct, Amplification, Polymerase Chain Reaction, FLAG-tag, Synthesized, Agarose Gel Electrophoresis, Transfection, Stable Transfection

    Real-time PCR analysis of viral DNA replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout bacmid or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.

    Journal: Virology

    Article Title: Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame

    doi: 10.1016/j.virol.2007.05.003

    Figure Lengend Snippet: Real-time PCR analysis of viral DNA replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout bacmid or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.

    Article Snippet: Bacmid DNA was purified from 0.5 L cultures using the plasmid Maxi plasmid DNA purification kit (Qiagen).

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Isolation, Knock-Out

    Clustering of C. difficile PCR-ribotypes. (A) Clustering of PRC-ribotypes based on fingerprinting profiles generated by capillary gel electrophoresis-based PCR-ribotyping. Dendrogram is color coded according to MLST type. The exact lengths of the bands, representing the 16S-23S rRNA intergenic spacer regions are given in Table S1 . (B) Minimum spanning tree of MLST results showing relatedness of PCR-ribotypes. Each circle represents one sequence type (ST) and is subdivided into sectors corresponding to the number of PCR-ribotypes represented with this ST. The numbers between circles represent number of differing loci between the STs.

    Journal: PLoS ONE

    Article Title: Recombination Drives Evolution of the Clostridium difficile 16S-23S rRNA Intergenic Spacer Region

    doi: 10.1371/journal.pone.0106545

    Figure Lengend Snippet: Clustering of C. difficile PCR-ribotypes. (A) Clustering of PRC-ribotypes based on fingerprinting profiles generated by capillary gel electrophoresis-based PCR-ribotyping. Dendrogram is color coded according to MLST type. The exact lengths of the bands, representing the 16S-23S rRNA intergenic spacer regions are given in Table S1 . (B) Minimum spanning tree of MLST results showing relatedness of PCR-ribotypes. Each circle represents one sequence type (ST) and is subdivided into sectors corresponding to the number of PCR-ribotypes represented with this ST. The numbers between circles represent number of differing loci between the STs.

    Article Snippet: Cloning and sequencing of 16S-23S rRNA ISR The purified amplification products were cloned into the pDrive vector (PCR Cloning Plus kit; Qiagen) according to the manufactureŕs instructions.

    Techniques: Polymerase Chain Reaction, Generated, Nucleic Acid Electrophoresis, Sequencing

    rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the PCR products that were amplified from the IL28A and IL28B promoters that included the SNP rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .

    Journal: PLoS ONE

    Article Title: A Single Nucleotide Polymorphism Associated with Hepatitis C Virus Infections Located in the Distal Region of the IL28B Promoter Influences NF-?B-Mediated Gene Transcription

    doi: 10.1371/journal.pone.0075495

    Figure Lengend Snippet: rs28416813 is in LD with rs12979860. A) EtBr-stained gel of the PCR products that were amplified from the IL28A and IL28B promoters that included the SNP rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends [35] . The ∼2 kb fragment specific to IL28B was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap [20] .

    Article Snippet: The SNP rs28416813 was amplified with the Long-range PCR kit from Qiagen by using: Forward primer 1.9kbrs813KpnIFor2 (5′GATATCGGTACCTGCATTGTACGACCCTCCAAC-3′) and reverse primer: 1.9kbil28b12aaHIIIREV1 (5′GATATCAAGCTTCAGCACTGCGGCCATCAG-3′).

    Techniques: Staining, Polymerase Chain Reaction, Amplification, Purification, DNA Sequencing, Marker, Generated

    ChIP-chip and ChIP-qPCR analysis of FoxP3 bound DNA from CD4 + CD25 hi human T reg cells. Anti-FoxP3 or control rabbit Ig was used to precipitate cross-linked protein–DNA complexes from expanded CD4 + CD25 hi human T reg cells lysate. The cross-linking of the immunoprecipitated material was removed and protease-treated and the DNA was purified and amplified. The resultant material was hybridized to the whole genome using GeneChip Human tiling 1.0R array set to identify the locations of binding sites for FoxP3. Two sets of graphs: FoxP3 IP versus the Ig control and FoxP3 IP versus Input DNA were generated on the hs.NCBIv35 version of the genome essentially following the method described in Cawley et el. (reference 50 ). (a) Signal enrichment graphs of IL-7R locus (chr5:35863179-35918811). Several regions in IL-7R locus are predicted to be positive (chr5:35892564-35892809 promoter) and negative (chr5:35890618-35890846 2K upstream; chr5:35907667-35907852 Intron 4; chr5:35911721-35911888 intron 7 and exon 8). (b) SYBR green qPCR of IL-7R chromosomal regions. FoxP3 IP versus the IgG fold enrichment ratio was determined from duplicate ChIP assay evaluated in duplicate by real time PCR.

    Journal: The Journal of Experimental Medicine

    Article Title: CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells

    doi: 10.1084/jem.20060772

    Figure Lengend Snippet: ChIP-chip and ChIP-qPCR analysis of FoxP3 bound DNA from CD4 + CD25 hi human T reg cells. Anti-FoxP3 or control rabbit Ig was used to precipitate cross-linked protein–DNA complexes from expanded CD4 + CD25 hi human T reg cells lysate. The cross-linking of the immunoprecipitated material was removed and protease-treated and the DNA was purified and amplified. The resultant material was hybridized to the whole genome using GeneChip Human tiling 1.0R array set to identify the locations of binding sites for FoxP3. Two sets of graphs: FoxP3 IP versus the Ig control and FoxP3 IP versus Input DNA were generated on the hs.NCBIv35 version of the genome essentially following the method described in Cawley et el. (reference 50 ). (a) Signal enrichment graphs of IL-7R locus (chr5:35863179-35918811). Several regions in IL-7R locus are predicted to be positive (chr5:35892564-35892809 promoter) and negative (chr5:35890618-35890846 2K upstream; chr5:35907667-35907852 Intron 4; chr5:35911721-35911888 intron 7 and exon 8). (b) SYBR green qPCR of IL-7R chromosomal regions. FoxP3 IP versus the IgG fold enrichment ratio was determined from duplicate ChIP assay evaluated in duplicate by real time PCR.

    Article Snippet: Gene expression was measured in real-time with the GeneAmp 7900 Sequence Detection System (Applied Biosystems) using primers and QuantiTect SYBR green PCR Kit purchased from QIAGEN.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Amplification, Binding Assay, Generated, SYBR Green Assay

    HIF1α is a key transcription factor upregulating HK2 expression in EML4-ALK-expressing lung cancer cells ( A ) The HK2 luciferase reporter pGL2-138HK2-Luc or the HRE mutant form was co-transfected into A549 cells along with pCDNA3.1/Zeo(+) (vector) or pCDNA3.1/Zeo(+)-EML4-ALK. The transactivation of the HK2 promoter by co-transfected ALK was measured by luciferase assays. The luciferase activities were presented as fold changes relative to the value of the vector control cells (defined as 1 fold). ChIP assay was performed to assess the binding of HIF1α to the HK2 promoter in ALK knockdown and control cells of H2228 and H3122 and in A549 cells transduced to express EML4-ALK. The HK2 promoter DNA sequence co-precipitated with HIF1α was analyzed by qPCR using SYBR Green ( B ) and regular PCR ( C ) using the primers listed in Table 1 . Fractions of immunoprecipitates and total cellular DNA were also analyzed with regular PCR amplification of the HK2 promoter..

    Journal: Oncogene

    Article Title: A causal link from ALK to hexokinase II overexpression and hyperactive glycolysis in EML4-ALK-positive lung cancer

    doi: 10.1038/onc.2016.150

    Figure Lengend Snippet: HIF1α is a key transcription factor upregulating HK2 expression in EML4-ALK-expressing lung cancer cells ( A ) The HK2 luciferase reporter pGL2-138HK2-Luc or the HRE mutant form was co-transfected into A549 cells along with pCDNA3.1/Zeo(+) (vector) or pCDNA3.1/Zeo(+)-EML4-ALK. The transactivation of the HK2 promoter by co-transfected ALK was measured by luciferase assays. The luciferase activities were presented as fold changes relative to the value of the vector control cells (defined as 1 fold). ChIP assay was performed to assess the binding of HIF1α to the HK2 promoter in ALK knockdown and control cells of H2228 and H3122 and in A549 cells transduced to express EML4-ALK. The HK2 promoter DNA sequence co-precipitated with HIF1α was analyzed by qPCR using SYBR Green ( B ) and regular PCR ( C ) using the primers listed in Table 1 . Fractions of immunoprecipitates and total cellular DNA were also analyzed with regular PCR amplification of the HK2 promoter..

    Article Snippet: The immunoprecipitates and input samples were reversed at 65°C overnight and then digested with Proteinase K. DNA samples were purified with the QIAquick Spin Columns (Qiagen, Valencia, CA) and quantified by qPCR with the QuantiNova SYBR Green PCR Kit (Qiagen) using primers in .

    Techniques: Expressing, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Amplification