pcr clean up kit  (Qiagen)

 
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    Name:
    QIAquick PCR Purification Kit
    Description:
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    Catalog Number:
    28104
    Price:
    117
    Category:
    QIAquick PCR Purification Kit
    Buy from Supplier


    Structured Review

    Qiagen pcr clean up kit
    QIAquick PCR Purification Kit
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    https://www.bioz.com/result/pcr clean up kit/product/Qiagen
    Average 99 stars, based on 194 article reviews
    Price from $9.99 to $1999.99
    pcr clean up kit - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination"

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

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0247-4

    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
    Figure Legend 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

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

    2) Product Images from "Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of the Congo"

    Article Title: Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of the Congo

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003212

    Amplicons generated with TbAT1 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45 to 48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.
    Figure Legend Snippet: Amplicons generated with TbAT1 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45 to 48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.

    Techniques Used: Generated, Polymerase Chain Reaction, Isolation

    Amplicons generated with AQP2/3 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45–48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.
    Figure Legend Snippet: Amplicons generated with AQP2/3 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45–48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.

    Techniques Used: Generated, Polymerase Chain Reaction, Isolation

    3) Product Images from "Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy"

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

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200419971

    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
    Figure Legend 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

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

    4) Product Images from "Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of the Congo"

    Article Title: Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of the Congo

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003212

    Amplicons generated with TbAT1 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45 to 48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.
    Figure Legend Snippet: Amplicons generated with TbAT1 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45 to 48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.

    Techniques Used: Generated, Polymerase Chain Reaction, Isolation

    Amplicons generated with AQP2/3 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45–48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.
    Figure Legend Snippet: Amplicons generated with AQP2/3 -PCR on DNA of the T.b. gambiense strains as listed in Table 1 and of the two T.b. brucei control strains. Lanes 1, 26, 27 and 52 = GeneRuler 100 bp Plus DNA Ladder (Fermentas), lanes 2 to 44 = T.b. gambiense strains isolated from Mbuji-Mayi, lanes 45–48 = T.b. gambiense strains isolated from Masi-Manimba, lane 49 = T.b. brucei 427 WT, lane 50 = T.b. brucei 427 AT1/P2 KO, lane 51 = negative PCR control.

    Techniques Used: Generated, Polymerase Chain Reaction, Isolation

    5) Product Images from "The Staphylococcal Accessory Regulator, SarA, is an RNA-Binding Protein that Modulates the mRNA Turnover Properties of Late-Exponential and Stationary Phase Staphylococcus aureus Cells"

    Article Title: The Staphylococcal Accessory Regulator, SarA, is an RNA-Binding Protein that Modulates the mRNA Turnover Properties of Late-Exponential and Stationary Phase Staphylococcus aureus Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2012.00026

    SarA binds mRNA in vivo . ( A) RT-PCR based detection of c-Myc-SarA transcript expression in Δ sarA and Δ sarA harboring plasmid pKLA40 harboring a c-Myc-SarA construct under control of the sarA P1 promoter during mid-exponential (ME), late-exponential (LE), and stationary (SP) phase growth. ( B) Western blotting based detection of c-Myc-SarA chimeric protein from sarA and Δ sarA harboring plasmid pKLA40; lysates collected during mid-exponential (ME), late-exponential (LE), and stationary (SP) phase growth. (C) SDS-PAGE and silver staining of exoproteins purified from wild type, Δ sarA and Δ sarA harboring plasmid pKLA40 stationary phase culture supernates. (D) RNA immunoprecipitation (RIP) was performed by capturing c-Myc-SarA protein cross-linked to nucleic acids. Successful immunoprecipitation was confirmed by Western blotting of cell lysate (L), lysate supernate (SN), wash (W), and elution (E) fractions with anti-c-Myc antibody.
    Figure Legend Snippet: SarA binds mRNA in vivo . ( A) RT-PCR based detection of c-Myc-SarA transcript expression in Δ sarA and Δ sarA harboring plasmid pKLA40 harboring a c-Myc-SarA construct under control of the sarA P1 promoter during mid-exponential (ME), late-exponential (LE), and stationary (SP) phase growth. ( B) Western blotting based detection of c-Myc-SarA chimeric protein from sarA and Δ sarA harboring plasmid pKLA40; lysates collected during mid-exponential (ME), late-exponential (LE), and stationary (SP) phase growth. (C) SDS-PAGE and silver staining of exoproteins purified from wild type, Δ sarA and Δ sarA harboring plasmid pKLA40 stationary phase culture supernates. (D) RNA immunoprecipitation (RIP) was performed by capturing c-Myc-SarA protein cross-linked to nucleic acids. Successful immunoprecipitation was confirmed by Western blotting of cell lysate (L), lysate supernate (SN), wash (W), and elution (E) fractions with anti-c-Myc antibody.

    Techniques Used: In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Construct, Western Blot, SDS Page, Silver Staining, Purification, Immunoprecipitation

    6) Product Images from "Analyses of Mitogenome Sequences Revealed that Asian Citrus Psyllids (Diaphorina citri) from California Were Related to Those from Florida"

    Article Title: Analyses of Mitogenome Sequences Revealed that Asian Citrus Psyllids (Diaphorina citri) from California Were Related to Those from Florida

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10713-3

    PCR and schematic nucleotide sequence alignments of the control region (CR) of Diaphorina citri . ( a ) Multiple amplicons of PCR of CR of individual D. citri from California, Florida and Guangdong-China based on primer set CR-f/CR-r. M, DNA Marker (2,000 bp); CApsy, FLpsy and GDpsy, D. citri from California, Florida and Guangdong-China, respectively, used for mitogenome sequencing; CA1-6: D. citri individuals from southern California, USA; FL1-6: D. citri individuals from Immokalee, Florida, USA; GD1-6: D. citri individuals from Guangzhou, Guangdong, China. BC, Bactericera cockerelli , showing a single amplicon from its CR as published previously 24 . ( b ) Schematic nucleotide sequence alignments of four variable amplicon clones from CR in a D. citri individual from California. rrnS = small ribosomal RNA.
    Figure Legend Snippet: PCR and schematic nucleotide sequence alignments of the control region (CR) of Diaphorina citri . ( a ) Multiple amplicons of PCR of CR of individual D. citri from California, Florida and Guangdong-China based on primer set CR-f/CR-r. M, DNA Marker (2,000 bp); CApsy, FLpsy and GDpsy, D. citri from California, Florida and Guangdong-China, respectively, used for mitogenome sequencing; CA1-6: D. citri individuals from southern California, USA; FL1-6: D. citri individuals from Immokalee, Florida, USA; GD1-6: D. citri individuals from Guangzhou, Guangdong, China. BC, Bactericera cockerelli , showing a single amplicon from its CR as published previously 24 . ( b ) Schematic nucleotide sequence alignments of four variable amplicon clones from CR in a D. citri individual from California. rrnS = small ribosomal RNA.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Marker, Amplification, Clone Assay

    7) Product Images from "Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins"

    Article Title: Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00859-z

    Autophagy/RELB suppress activation of TGFβ gene promoters independent of NF-κB binding elements. a WT, Δ ATG5 and Δ RELB A549 cells were subjected to chromatin immunoprecipitation (ChIP) for dimethyl-K4-histone H3 (H3Me2K4). Precipitating proximal promoter DNA was quantified by qRT-PCR, expressed as a percentage of input (means, n = 3, ±S.D., * P
    Figure Legend Snippet: Autophagy/RELB suppress activation of TGFβ gene promoters independent of NF-κB binding elements. a WT, Δ ATG5 and Δ RELB A549 cells were subjected to chromatin immunoprecipitation (ChIP) for dimethyl-K4-histone H3 (H3Me2K4). Precipitating proximal promoter DNA was quantified by qRT-PCR, expressed as a percentage of input (means, n = 3, ±S.D., * P

    Techniques Used: Activation Assay, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR

    Autophagy promotes tumorigenesis in vivo. a A549 cells deficient in ATG5 protein expression were generated by CRISPR/Cas9-mediated genome editing. Each of the two wild-type control clones (WT and WT-2) and Δ ATG5 clones (Δ ATG5 and Δ ATG5 -2) were immunoblotted for the indicated proteins. b Genomic DNA from A549 Δ ATG5 and Δ ATG5 -2 cells was PCR amplified. The amplicon encompassed the sequence to which Cas9 had been targeted (PAM in bold ) and different indels were identified in both clones via Sanger sequencing. c A549 WT and Δ ATG5 cells were plated at low confluency for time-lapse phase-contrast videomicroscopy using an Incucyte microscope and cell proliferation was monitored by automated confluency analysis at set intervals post plating (means, n = 9 wells, ±S.D.). d A pooled derivative of Δ ATG5 cells was generated by stable transduction with GFP-ATG5 retrovirus (rescue). The indicated cell lines were immunoblotted as shown. e WT, Δ ATG5 and rescue cells were subcutaneously injected into immunocompromised mice and tumour volume was measured longitudinally (means, n = 10 flanks, ±S.E.M., * P
    Figure Legend Snippet: Autophagy promotes tumorigenesis in vivo. a A549 cells deficient in ATG5 protein expression were generated by CRISPR/Cas9-mediated genome editing. Each of the two wild-type control clones (WT and WT-2) and Δ ATG5 clones (Δ ATG5 and Δ ATG5 -2) were immunoblotted for the indicated proteins. b Genomic DNA from A549 Δ ATG5 and Δ ATG5 -2 cells was PCR amplified. The amplicon encompassed the sequence to which Cas9 had been targeted (PAM in bold ) and different indels were identified in both clones via Sanger sequencing. c A549 WT and Δ ATG5 cells were plated at low confluency for time-lapse phase-contrast videomicroscopy using an Incucyte microscope and cell proliferation was monitored by automated confluency analysis at set intervals post plating (means, n = 9 wells, ±S.D.). d A pooled derivative of Δ ATG5 cells was generated by stable transduction with GFP-ATG5 retrovirus (rescue). The indicated cell lines were immunoblotted as shown. e WT, Δ ATG5 and rescue cells were subcutaneously injected into immunocompromised mice and tumour volume was measured longitudinally (means, n = 10 flanks, ±S.E.M., * P

    Techniques Used: In Vivo, Expressing, Generated, CRISPR, Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing, Microscopy, Transduction, Injection, Mouse Assay

    8) Product Images from "A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays"

    Article Title: A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-10-497

    Specific DNA binding of HaloTag-CREB in vivo . HaloCHIP experiments were performed in triplicates, on HeLa cells transiently expressing HaloTag-CREB or untransfected as a control. Resulting DNA from both the HaloTag-CREB and Untransfected control sample was amplified and analyzed using Plexor quantitative PCR. Total amounts of DNA for both samples were calculated for three promoters which CREB is known to bind [ 28 ], Fos, Jun, and p27, as well as three control sequences, C1, C2, and C3, which do not contain CRE consensus binding sites. Depicted in dark blue is the fold enrichment of each CREB-specific promoter over the average amount of the three control promoters for the HaloTag-CREB HaloCHIP experimental sample. In light blue is the identical calculation for the Untransfected HaloCHIP control sample.
    Figure Legend Snippet: Specific DNA binding of HaloTag-CREB in vivo . HaloCHIP experiments were performed in triplicates, on HeLa cells transiently expressing HaloTag-CREB or untransfected as a control. Resulting DNA from both the HaloTag-CREB and Untransfected control sample was amplified and analyzed using Plexor quantitative PCR. Total amounts of DNA for both samples were calculated for three promoters which CREB is known to bind [ 28 ], Fos, Jun, and p27, as well as three control sequences, C1, C2, and C3, which do not contain CRE consensus binding sites. Depicted in dark blue is the fold enrichment of each CREB-specific promoter over the average amount of the three control promoters for the HaloTag-CREB HaloCHIP experimental sample. In light blue is the identical calculation for the Untransfected HaloCHIP control sample.

    Techniques Used: Binding Assay, In Vivo, Expressing, Amplification, Real-time Polymerase Chain Reaction

    9) Product Images from "A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays"

    Article Title: A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-10-497

    Specific DNA binding of HaloTag-CREB in vivo . HaloCHIP experiments were performed in triplicates, on HeLa cells transiently expressing HaloTag-CREB or untransfected as a control. Resulting DNA from both the HaloTag-CREB and Untransfected control sample was amplified and analyzed using Plexor quantitative PCR. Total amounts of DNA for both samples were calculated for three promoters which CREB is known to bind [ 28 ], Fos, Jun, and p27, as well as three control sequences, C1, C2, and C3, which do not contain CRE consensus binding sites. Depicted in dark blue is the fold enrichment of each CREB-specific promoter over the average amount of the three control promoters for the HaloTag-CREB HaloCHIP experimental sample. In light blue is the identical calculation for the Untransfected HaloCHIP control sample.
    Figure Legend Snippet: Specific DNA binding of HaloTag-CREB in vivo . HaloCHIP experiments were performed in triplicates, on HeLa cells transiently expressing HaloTag-CREB or untransfected as a control. Resulting DNA from both the HaloTag-CREB and Untransfected control sample was amplified and analyzed using Plexor quantitative PCR. Total amounts of DNA for both samples were calculated for three promoters which CREB is known to bind [ 28 ], Fos, Jun, and p27, as well as three control sequences, C1, C2, and C3, which do not contain CRE consensus binding sites. Depicted in dark blue is the fold enrichment of each CREB-specific promoter over the average amount of the three control promoters for the HaloTag-CREB HaloCHIP experimental sample. In light blue is the identical calculation for the Untransfected HaloCHIP control sample.

    Techniques Used: Binding Assay, In Vivo, Expressing, Amplification, Real-time Polymerase Chain Reaction

    10) Product Images from "PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination"

    Article Title: PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004916

    Novel hotspots have biased H3K4me3 modification. (A) F1 hybrid hotspots were classified as B6 or CAST depending on parental origin, or labeled novel. (B) H3K4me3 haplotype ratio for BxC TSS (n = 12,903, orange - autosomes, black - chromosome X). (C) H3K4me3 haplotype ratio for BxC hotspots (grey - all BxC hotspots, n = 12,271; green - novel BxC hotspots, n = 2,298). (D) Scatterplot of haplotype-specific H3K4me3 for hotspots shared between progeny from reciprocal B6 and CAST crosses (n = 10,977, r = 0.978 without X chromosome). 163 hotspots in lower right (circle) are all on the chromosome X. Black shading reflects signal density. (E) Left - Haplotype-specific PCR showing from genomic DNA samples for hotspots chr1 185.33 Mb and chr1 171.37 Mb. Right - Haplotype-specific PCR from spermatocytes before or after enrichment for H3K4me3 (n = 4, error bars - S.D.). (F) The parental identity and fraction of hotpots in WxP F1 hybrids. (G) H3K4me3 haplotype ratio for WxP TSS (n = 15,856, orange - autosomes, black - chromosome X). (H) H3K4me3 haplotype ratio for WxP hotspots (grey - all WxP hotspots, n = 8,360; green - novel WxP hotspots, n = 2,325).
    Figure Legend Snippet: Novel hotspots have biased H3K4me3 modification. (A) F1 hybrid hotspots were classified as B6 or CAST depending on parental origin, or labeled novel. (B) H3K4me3 haplotype ratio for BxC TSS (n = 12,903, orange - autosomes, black - chromosome X). (C) H3K4me3 haplotype ratio for BxC hotspots (grey - all BxC hotspots, n = 12,271; green - novel BxC hotspots, n = 2,298). (D) Scatterplot of haplotype-specific H3K4me3 for hotspots shared between progeny from reciprocal B6 and CAST crosses (n = 10,977, r = 0.978 without X chromosome). 163 hotspots in lower right (circle) are all on the chromosome X. Black shading reflects signal density. (E) Left - Haplotype-specific PCR showing from genomic DNA samples for hotspots chr1 185.33 Mb and chr1 171.37 Mb. Right - Haplotype-specific PCR from spermatocytes before or after enrichment for H3K4me3 (n = 4, error bars - S.D.). (F) The parental identity and fraction of hotpots in WxP F1 hybrids. (G) H3K4me3 haplotype ratio for WxP TSS (n = 15,856, orange - autosomes, black - chromosome X). (H) H3K4me3 haplotype ratio for WxP hotspots (grey - all WxP hotspots, n = 8,360; green - novel WxP hotspots, n = 2,325).

    Techniques Used: Modification, Labeling, Polymerase Chain Reaction

    11) Product Images from "AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication"

    Article Title: AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097579

    ZFN-induced target site disruption in HepAD38 cells. ( a, b ) Cells were transduced with scAAV2 vectors expressing ZFN pairs 1, 2, 3 or all three pairs together (1–3) at a total MOI of 10000 genomes/cell. The presence of mutations was analyzed in DNA isolated from transduced cells 72 hours later using the Surveyor nuclease assay. ( c ) For ZFN site 3, an analysis of DNA mutagenic events disrupting the internal NcoI cleavage site was also performed. Above the gel images, the sizes of PCR amplicons and the cleavage products produced upon Surveyor nuclease cleavage (indicating mutations at the indicated target site) or NcoI cleavage are shown. bp – base pairs; UC – untreated control; ZFN – zinc finger nuclease. Bands indicating mutations are highlighted with an asterisk and the percentage of ZFN-mediated DNA mutation for each targeted site is indicated. ( d ) DNA mutations that were detected at ZFN target sites 1, 2 and 3 within HepAD38 HBV sequences are shown above the wild-type ZFN site. Nucleotides with differences in at least one sequence are shown in color. Spacer regions are underlined. The rates at which DNA mutations were detected are listed in Table 1 . wt – wild type.
    Figure Legend Snippet: ZFN-induced target site disruption in HepAD38 cells. ( a, b ) Cells were transduced with scAAV2 vectors expressing ZFN pairs 1, 2, 3 or all three pairs together (1–3) at a total MOI of 10000 genomes/cell. The presence of mutations was analyzed in DNA isolated from transduced cells 72 hours later using the Surveyor nuclease assay. ( c ) For ZFN site 3, an analysis of DNA mutagenic events disrupting the internal NcoI cleavage site was also performed. Above the gel images, the sizes of PCR amplicons and the cleavage products produced upon Surveyor nuclease cleavage (indicating mutations at the indicated target site) or NcoI cleavage are shown. bp – base pairs; UC – untreated control; ZFN – zinc finger nuclease. Bands indicating mutations are highlighted with an asterisk and the percentage of ZFN-mediated DNA mutation for each targeted site is indicated. ( d ) DNA mutations that were detected at ZFN target sites 1, 2 and 3 within HepAD38 HBV sequences are shown above the wild-type ZFN site. Nucleotides with differences in at least one sequence are shown in color. Spacer regions are underlined. The rates at which DNA mutations were detected are listed in Table 1 . wt – wild type.

    Techniques Used: Transduction, Expressing, Isolation, Nuclease Assay, Polymerase Chain Reaction, Produced, Zinc-Fingers, Mutagenesis, Sequencing

    12) Product Images from "Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination"

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

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0247-4

    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
    Figure Legend 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

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

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    Transfection:

    Article Title: Epigenetic Regulation of HYAL-1 Hyaluronidase Expression
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    Purification:

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

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    Immunoprecipitation:

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

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    Polymerase Chain Reaction:

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination
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  • 99
    Qiagen pcr 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
    Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up kit/product/Qiagen
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    pcr clean up kit - by Bioz Stars, 2020-07
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    Qiagen 96 well plates multichannel pipettor optional pcr clean up kit
    Examples of a saw-tooth pattern in agarose gel analysis of <t>PCR</t> product. PCR mixtures (96 samples) were run on a 1% agarose gel. A primer-ordering algorithm generates a map of oligonucleotide primers in <t>96-well</t> format with alternating expected product
    96 Well Plates Multichannel Pipettor Optional Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/96 well plates multichannel pipettor optional pcr clean up kit/product/Qiagen
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    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

    Examples of a saw-tooth pattern in agarose gel analysis of PCR product. PCR mixtures (96 samples) were run on a 1% agarose gel. A primer-ordering algorithm generates a map of oligonucleotide primers in 96-well format with alternating expected product

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

    doi: 10.1002/0471142727.mb0320s110

    Figure Lengend Snippet: Examples of a saw-tooth pattern in agarose gel analysis of PCR product. PCR mixtures (96 samples) were run on a 1% agarose gel. A primer-ordering algorithm generates a map of oligonucleotide primers in 96-well format with alternating expected product

    Article Snippet: 10 mM 4dNTPs 100% DMSO 2.0 U/µl Phusion® High-Fidelity DNA polymerase (New England BioLabs (NEB)) ( Most high-fidelity polymerases should be sufficient ) 5× Phusion® HF Buffer (NEB) 2× CloneAmp™ HiFi PCR Premix (Clontech) DNA template: 10 ng/µl first-strand cDNA mix or 1–50 ng/µl plasmid DNA 10 µM gene specific 5′-oligonucleotide primer in sterile H2 O (for primer design see Basic Protocol 1 introduction) 10 µM gene specific 3′-oligonucleotide primer in sterile H2 O (for primer design see Basic Protocol 1 introduction) 100 µM universal 5′-oligonucleotide primer (Gateway® only) 100 µM universal 3′-oligonucleotide primer (Gateway® only) PCR tubes (thin-walled) or 96-well plates Multichannel pipettor (optional) PCR Clean-Up kit (e.g., Qiagen or Macherey Nagel for high-throughput) Centrifuge Thermal cycler Additional reagents and equipment for gel electrophoresis (see unit 2.5A ) and DNA preparation (see unit 1.6 or commercial DNA prep kit).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction