pcr cleanup kit  (Qiagen)

 
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    MinElute Reaction Cleanup Kit
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    For cleanup of up to 5 μg DNA 70 bp to 4 kb from enzymatic reactions Kit contents Qiagen minElute Reaction Cleanup Kit 50 preps 10L Elution Volume 5g Binding Capacity Manual Processing Silica Technology Tube Format 70 bp to 4 kb Fragment Size Provides Spin Columns Fast Procedure and Easy Handling Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion and Labeling For Cleanup of up to 5μg DNA from Enzymatic Reactions Includes 50 minElute Spin Columns Buffers Collection Tubes 2mL Benefits Very small elution volumes Fast procedure and easy handling High reproducible recoveries Gel loading dye for convenient sample analysis
    Catalog Number:
    28204
    Price:
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    MinElute Reaction Cleanup Kit
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    Structured Review

    Qiagen pcr cleanup kit
    MinElute Reaction Cleanup Kit
    For cleanup of up to 5 μg DNA 70 bp to 4 kb from enzymatic reactions Kit contents Qiagen minElute Reaction Cleanup Kit 50 preps 10L Elution Volume 5g Binding Capacity Manual Processing Silica Technology Tube Format 70 bp to 4 kb Fragment Size Provides Spin Columns Fast Procedure and Easy Handling Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion and Labeling For Cleanup of up to 5μg DNA from Enzymatic Reactions Includes 50 minElute Spin Columns Buffers Collection Tubes 2mL Benefits Very small elution volumes Fast procedure and easy handling High reproducible recoveries Gel loading dye for convenient sample analysis
    https://www.bioz.com/result/pcr cleanup kit/product/Qiagen
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    Price from $9.99 to $1999.99
    pcr cleanup kit - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Initiation of Early Osteoblast Differentiation Events through the Direct Transcriptional Regulation of Msx2 by FOXC1"

    Article Title: Initiation of Early Osteoblast Differentiation Events through the Direct Transcriptional Regulation of Msx2 by FOXC1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049095

    FOXC1 binds to the Msx2 promoter in vivo. (A) Sequence analysis of upstream regulatory elements reveals the presence of a FOXC1 binding motif, indicated in bold, (TAAAT/CAAT) located in a conserved motif near the predicted Msx2 transcription start site of the mouse, rat and human genes. Small arrows correspond to the position of ChIP primers located in the promoter region or the coding region of mouse Msx2 . Nucleotide sequences of the Electrophoretic mobility shift assay (EMSA) probes for wild type (WT) and mutated (MUT) FOXC1 binding sites are indicated. (B) Chromatin immunoprecipitation assays confirm the binding of FOXC1 to the Msx2 promoter in vivo . Quantitative PCR (qPCR) was conducted on ChiP products isolated from 10T1/2 cells using antibodies recognizing FOXC1 or normal immunoglobulins (IgG). Primers were designed amplify regions in the promoter flanking the putative FOXC1 binding site or exon 2 of the mouse Msx2 gene. Amplification signals are presented a percentage compared to input chromatin fraction. (C) EMSAs demonstrate FOXC1 binding to DNA elements in the Msx2 promoter. Extracts from U2OS cells or cells transfected with FOXC1 were incubated with IR700-labeled oligonucleotides correspond to the WT or MUT FOXC1 binding sites. FOXC1-DNA complexes are indicated by the arrow.
    Figure Legend Snippet: FOXC1 binds to the Msx2 promoter in vivo. (A) Sequence analysis of upstream regulatory elements reveals the presence of a FOXC1 binding motif, indicated in bold, (TAAAT/CAAT) located in a conserved motif near the predicted Msx2 transcription start site of the mouse, rat and human genes. Small arrows correspond to the position of ChIP primers located in the promoter region or the coding region of mouse Msx2 . Nucleotide sequences of the Electrophoretic mobility shift assay (EMSA) probes for wild type (WT) and mutated (MUT) FOXC1 binding sites are indicated. (B) Chromatin immunoprecipitation assays confirm the binding of FOXC1 to the Msx2 promoter in vivo . Quantitative PCR (qPCR) was conducted on ChiP products isolated from 10T1/2 cells using antibodies recognizing FOXC1 or normal immunoglobulins (IgG). Primers were designed amplify regions in the promoter flanking the putative FOXC1 binding site or exon 2 of the mouse Msx2 gene. Amplification signals are presented a percentage compared to input chromatin fraction. (C) EMSAs demonstrate FOXC1 binding to DNA elements in the Msx2 promoter. Extracts from U2OS cells or cells transfected with FOXC1 were incubated with IR700-labeled oligonucleotides correspond to the WT or MUT FOXC1 binding sites. FOXC1-DNA complexes are indicated by the arrow.

    Techniques Used: In Vivo, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Electrophoretic Mobility Shift Assay, Real-time Polymerase Chain Reaction, Isolation, Amplification, Transfection, Incubation, Labeling

    2) Product Images from "Chromatin restriction by the nucleosome remodeler Mi-2β and functional interplay with lineage-specific transcription regulators control B-cell differentiation"

    Article Title: Chromatin restriction by the nucleosome remodeler Mi-2β and functional interplay with lineage-specific transcription regulators control B-cell differentiation

    Journal: Genes & Development

    doi: 10.1101/gad.321901.118

    Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative RT-PCR analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic DNA samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.
    Figure Legend Snippet: Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative RT-PCR analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic DNA samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Expressing, Polymerase Chain Reaction, Amplification, Southern Blot, Flow Cytometry, Cytometry, Cell Differentiation

    3) Product Images from "ETO/MTG8 Is an Inhibitor of C/EBP? Activity and a Regulator of Early Adipogenesis"

    Article Title: ETO/MTG8 Is an Inhibitor of C/EBP? Activity and a Regulator of Early Adipogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9863-9872.2004

    ETO interacts directly with C/EBPβ inhibiting its DNA-binding activity toward the C/EBPα promoter and preventing centromeric localization during adipogenesis. (A) HEK293 cells were transfected with control vector, GFP-ETO (G-ETO-WT), or GFP-ETO-AA in the absence or presence of C/EBPβ as indicated. Anti-C/EBPβ immunoprecipitates were analyzed for associated ETO protein (upper panel), whereas corresponding cell lysates were probed for ETO (middle panel) or C/EBPβ (lower panel) by Western blotting. (B) 3T3-L1 preadipocytes expressing GFP-ETO were treated for 4 h in the absence or presence of differentiation cocktail as indicated prior to lysis. Cell lysates (left panels) or immunoprecipitates prepared by using an anti-ETO antibody (Santa Cruz Biotechnology) (right panels) were analyzed by Western blotting to detect ETO (upper panels) or C/EBPβ isoforms (lower panels). (C) In vitro transcribed/translated C/EBPβ (Cβ) and/or ETO (E) were incubated with radiolabeled DNA probe corresponding to the proximal C/EBPα binding site of the C/EBPα promoter in a gel shift assay. Various ratios of C/EBPβ to ETO were achieved by adjusting the quantity of ETO. In all lanes total protein input was kept constant by appropriate addition of rabbit reticulocyte lysate, except in lane 1, where free labeled probe was run alone. (D) 3T3-L1 preadipocytes were induced to differentiate for the times indicated and ChIP assays performed by using an anti-C/EBPβ antibody to isolate C/EBPβ-associated DNA. DNA from these immunoprecipitates corresponding to the C/EBPβ binding site in the C/EBPα promoter was quantified by using real-time PCR and normalized to DNA from a 10% sample of corresponding input lysate. The data are means ± the SEM from three independent experiments. Asterisks indicate a statistically significant difference ( P
    Figure Legend Snippet: ETO interacts directly with C/EBPβ inhibiting its DNA-binding activity toward the C/EBPα promoter and preventing centromeric localization during adipogenesis. (A) HEK293 cells were transfected with control vector, GFP-ETO (G-ETO-WT), or GFP-ETO-AA in the absence or presence of C/EBPβ as indicated. Anti-C/EBPβ immunoprecipitates were analyzed for associated ETO protein (upper panel), whereas corresponding cell lysates were probed for ETO (middle panel) or C/EBPβ (lower panel) by Western blotting. (B) 3T3-L1 preadipocytes expressing GFP-ETO were treated for 4 h in the absence or presence of differentiation cocktail as indicated prior to lysis. Cell lysates (left panels) or immunoprecipitates prepared by using an anti-ETO antibody (Santa Cruz Biotechnology) (right panels) were analyzed by Western blotting to detect ETO (upper panels) or C/EBPβ isoforms (lower panels). (C) In vitro transcribed/translated C/EBPβ (Cβ) and/or ETO (E) were incubated with radiolabeled DNA probe corresponding to the proximal C/EBPα binding site of the C/EBPα promoter in a gel shift assay. Various ratios of C/EBPβ to ETO were achieved by adjusting the quantity of ETO. In all lanes total protein input was kept constant by appropriate addition of rabbit reticulocyte lysate, except in lane 1, where free labeled probe was run alone. (D) 3T3-L1 preadipocytes were induced to differentiate for the times indicated and ChIP assays performed by using an anti-C/EBPβ antibody to isolate C/EBPβ-associated DNA. DNA from these immunoprecipitates corresponding to the C/EBPβ binding site in the C/EBPα promoter was quantified by using real-time PCR and normalized to DNA from a 10% sample of corresponding input lysate. The data are means ± the SEM from three independent experiments. Asterisks indicate a statistically significant difference ( P

    Techniques Used: Binding Assay, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Lysis, In Vitro, Incubation, Electrophoretic Mobility Shift Assay, Labeling, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    4) Product Images from "A Novel Group of Moraxella catarrhalis UspA Proteins Mediates Cellular Adhesion via CEACAMs and Vitronectin"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045452

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Polymerase Chain Reaction

    5) Product Images from "RNA aptamers selected against DNA polymerase ? inhibit the polymerase activities of DNA polymerases ? and ?"

    Article Title: RNA aptamers selected against DNA polymerase ? inhibit the polymerase activities of DNA polymerases ? and ?

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl326

    Sequences and structure of selected aptamers. (Upper) Sequences of the primers, the RNA pool and the polβ aptamers. The PCR primer annealing sites within the constant regions are underlined. For the RNA library, the randomized region (n30) is flanked by the 5′ and 3′ constant regions. The names of the representative aptamers are shown on the left. The number in parentheses indicates the number of independent clones of the identical sequence. In aptamer 45, the last C in the 5′ constant region has been mutated to a U. The sequences are shown with the most similar sequences grouped together according to Clustal ( 49 ). (Lower) The Mfold program (available at ) was used to derive secondary structural models for aptamers 25 (−17.6 kcal/mol, next most stable −17.0 kcal/mol), 31 (−15.3 kcal/mol, next most stable −14.5 kcal/mol), 32 (−19.3 kcal/mol, next most stable −18.3 kcal/mol), 45 (−21.2 kcal/mol, next most stable −20.6 kcal/mol) and 53 (−20.2 kcal/mol, next most stable −19.4 kcal/mol). Only the most stable predicted structure is shown.
    Figure Legend Snippet: Sequences and structure of selected aptamers. (Upper) Sequences of the primers, the RNA pool and the polβ aptamers. The PCR primer annealing sites within the constant regions are underlined. For the RNA library, the randomized region (n30) is flanked by the 5′ and 3′ constant regions. The names of the representative aptamers are shown on the left. The number in parentheses indicates the number of independent clones of the identical sequence. In aptamer 45, the last C in the 5′ constant region has been mutated to a U. The sequences are shown with the most similar sequences grouped together according to Clustal ( 49 ). (Lower) The Mfold program (available at ) was used to derive secondary structural models for aptamers 25 (−17.6 kcal/mol, next most stable −17.0 kcal/mol), 31 (−15.3 kcal/mol, next most stable −14.5 kcal/mol), 32 (−19.3 kcal/mol, next most stable −18.3 kcal/mol), 45 (−21.2 kcal/mol, next most stable −20.6 kcal/mol) and 53 (−20.2 kcal/mol, next most stable −19.4 kcal/mol). Only the most stable predicted structure is shown.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Sequencing

    6) Product Images from "SMYD3 Promotes Homologous Recombination via Regulation of H3K4-mediated Gene Expression"

    Article Title: SMYD3 Promotes Homologous Recombination via Regulation of H3K4-mediated Gene Expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03385-6

    SMYD3 knockdown downregulates HR gene expressions. ( a ) The confirmation of the microarray analyses for the expressions of candidate genes in the shSMYD3/shLuc dataset using qRT-PCR. The fold changes of each gene expression in the microarray data were listed below. * P
    Figure Legend Snippet: SMYD3 knockdown downregulates HR gene expressions. ( a ) The confirmation of the microarray analyses for the expressions of candidate genes in the shSMYD3/shLuc dataset using qRT-PCR. The fold changes of each gene expression in the microarray data were listed below. * P

    Techniques Used: Microarray, Quantitative RT-PCR, Expressing

    SMYD3 regulates the expression of MDC1 through methylating histone H3K4. ( a ) ChIP assay was performed in MCF7 cells using specific antibodies. The examined positions at the MDC1 locus were indicated, in which region S3 and region TA for the predicted SMYD3 and TATA box binding sites, respectively. ( b – e ) ChIP assays were performed with SMYD3-repressed MCF7 cells using specific antibodies shown at the top of the histogram. Fold enrichment of each antibody compared with IgG was shown. ( f ) Ratios of H3K4me3/H3 ChIP signals were displayed. In ( b – f ), immunoprecipitated chromatin was quantified by qRT-PCR. * P
    Figure Legend Snippet: SMYD3 regulates the expression of MDC1 through methylating histone H3K4. ( a ) ChIP assay was performed in MCF7 cells using specific antibodies. The examined positions at the MDC1 locus were indicated, in which region S3 and region TA for the predicted SMYD3 and TATA box binding sites, respectively. ( b – e ) ChIP assays were performed with SMYD3-repressed MCF7 cells using specific antibodies shown at the top of the histogram. Fold enrichment of each antibody compared with IgG was shown. ( f ) Ratios of H3K4me3/H3 ChIP signals were displayed. In ( b – f ), immunoprecipitated chromatin was quantified by qRT-PCR. * P

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

    7) Product Images from "RNA aptamers selected against DNA polymerase ? inhibit the polymerase activities of DNA polymerases ? and ?"

    Article Title: RNA aptamers selected against DNA polymerase ? inhibit the polymerase activities of DNA polymerases ? and ?

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl326

    Sequences and structure of selected aptamers. (Upper) Sequences of the primers, the RNA pool and the polβ aptamers. The PCR primer annealing sites within the constant regions are underlined. For the RNA library, the randomized region (n30) is flanked by the 5′ and 3′ constant regions. The names of the representative aptamers are shown on the left. The number in parentheses indicates the number of independent clones of the identical sequence. In aptamer 45, the last C in the 5′ constant region has been mutated to a U. The sequences are shown with the most similar sequences grouped together according to Clustal ( 49 ). (Lower) The Mfold program (available at ) was used to derive secondary structural models for aptamers 25 (−17.6 kcal/mol, next most stable −17.0 kcal/mol), 31 (−15.3 kcal/mol, next most stable −14.5 kcal/mol), 32 (−19.3 kcal/mol, next most stable −18.3 kcal/mol), 45 (−21.2 kcal/mol, next most stable −20.6 kcal/mol) and 53 (−20.2 kcal/mol, next most stable −19.4 kcal/mol). Only the most stable predicted structure is shown.
    Figure Legend Snippet: Sequences and structure of selected aptamers. (Upper) Sequences of the primers, the RNA pool and the polβ aptamers. The PCR primer annealing sites within the constant regions are underlined. For the RNA library, the randomized region (n30) is flanked by the 5′ and 3′ constant regions. The names of the representative aptamers are shown on the left. The number in parentheses indicates the number of independent clones of the identical sequence. In aptamer 45, the last C in the 5′ constant region has been mutated to a U. The sequences are shown with the most similar sequences grouped together according to Clustal ( 49 ). (Lower) The Mfold program (available at ) was used to derive secondary structural models for aptamers 25 (−17.6 kcal/mol, next most stable −17.0 kcal/mol), 31 (−15.3 kcal/mol, next most stable −14.5 kcal/mol), 32 (−19.3 kcal/mol, next most stable −18.3 kcal/mol), 45 (−21.2 kcal/mol, next most stable −20.6 kcal/mol) and 53 (−20.2 kcal/mol, next most stable −19.4 kcal/mol). Only the most stable predicted structure is shown.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Sequencing

    8) Product Images from "Tropomyosin isoforms differentially affect muscle contractility in the head and body regions of the nematode Caenorhabditis elegans"

    Article Title: Tropomyosin isoforms differentially affect muscle contractility in the head and body regions of the nematode Caenorhabditis elegans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-03-0152

    Characterization of mutually exclusive seventh exons of the C. elegans lev-11 tropomyosin gene. (A) Structure of the lev-11 gene is shown schematically (top) with numbered boxes indicating exons. Recently characterized alternative exons 7a and 7b are shown in green. Below the gene structure are splicing patterns of LEV-11A/CeTMI, a newly identified isoform, LEV-11O, and three previously characterized isoforms, LEV-11D/CeTMII, LEV-11E/CeTMIII, and LEV-11C/CeTMIV. Coding and noncoding regions are shown in orange and light yellow, respectively. Note that exon 9c is used in all known isoforms either as a coding region (LEV-11A and LEV-11O) or as a noncoding region when 9a or 9b is used as a coding region (LEV-11C, LEV-11D, and LEV-11E). (B) Analysis of lev-11 mRNAs by RT-PCR. Total RNAs from synchronized wild-type L1 larvae were subjected to RT-PCR with indicated primer pairs and cycle numbers, and the PCR products were analyzed with a 2100 BioAnalyzer (Agilent). DNA size markers are shown on the left. Results are shown in gel-like presentations. Exon combinations of representative bands a–d are shown below. Multiple bands in E7b/E9c (lane 6) were cloned and sequenced, and the exon combinations are indicated on the right. Asterisks indicate artificial PCR products due to excessive cycles. (C) Alignment of amino acid sequences encoded by exons 7a and 7b. Identical residues are indicated with black backgrounds. Point mutations in lev-11(gk334531) and lev-11(x12) are shown at the top and bottom of the sequences, respectively. (D) Probability of coiled-coil formation (0–1) was calculated from the full-length sequences of LEV-11A and LEV-11O by COILS ( Lupas et al. , 1991 ), and plots of exon 7-coded regions are shown.
    Figure Legend Snippet: Characterization of mutually exclusive seventh exons of the C. elegans lev-11 tropomyosin gene. (A) Structure of the lev-11 gene is shown schematically (top) with numbered boxes indicating exons. Recently characterized alternative exons 7a and 7b are shown in green. Below the gene structure are splicing patterns of LEV-11A/CeTMI, a newly identified isoform, LEV-11O, and three previously characterized isoforms, LEV-11D/CeTMII, LEV-11E/CeTMIII, and LEV-11C/CeTMIV. Coding and noncoding regions are shown in orange and light yellow, respectively. Note that exon 9c is used in all known isoforms either as a coding region (LEV-11A and LEV-11O) or as a noncoding region when 9a or 9b is used as a coding region (LEV-11C, LEV-11D, and LEV-11E). (B) Analysis of lev-11 mRNAs by RT-PCR. Total RNAs from synchronized wild-type L1 larvae were subjected to RT-PCR with indicated primer pairs and cycle numbers, and the PCR products were analyzed with a 2100 BioAnalyzer (Agilent). DNA size markers are shown on the left. Results are shown in gel-like presentations. Exon combinations of representative bands a–d are shown below. Multiple bands in E7b/E9c (lane 6) were cloned and sequenced, and the exon combinations are indicated on the right. Asterisks indicate artificial PCR products due to excessive cycles. (C) Alignment of amino acid sequences encoded by exons 7a and 7b. Identical residues are indicated with black backgrounds. Point mutations in lev-11(gk334531) and lev-11(x12) are shown at the top and bottom of the sequences, respectively. (D) Probability of coiled-coil formation (0–1) was calculated from the full-length sequences of LEV-11A and LEV-11O by COILS ( Lupas et al. , 1991 ), and plots of exon 7-coded regions are shown.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay

    9) Product Images from "Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor-DNA Interactions"

    Article Title: Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor-DNA Interactions

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000449

    Dot6 binds to PAC-containing promoters in vivo. Chromatin immunoprecipitation was performed on extracts of strain Y3648 (TAP-Dot6) and B4741 (untagged) as described in Materials and Methods . Quantification of these data provided the percent of input DNA for the promoters of each of the indicated genes recovered in the immunoprecipitate. Shown are the fold enrichment of those values relative to the percent input DNA for the ACT1 promoter recovered in the same immunoprecipitate. PCR quantification was performed in triplicate with less than 20% variation among replicates of individual samples.
    Figure Legend Snippet: Dot6 binds to PAC-containing promoters in vivo. Chromatin immunoprecipitation was performed on extracts of strain Y3648 (TAP-Dot6) and B4741 (untagged) as described in Materials and Methods . Quantification of these data provided the percent of input DNA for the promoters of each of the indicated genes recovered in the immunoprecipitate. Shown are the fold enrichment of those values relative to the percent input DNA for the ACT1 promoter recovered in the same immunoprecipitate. PCR quantification was performed in triplicate with less than 20% variation among replicates of individual samples.

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    An overview of Microarray profiling of phage-display selection technology. (A) 1–3 kb fragments of yeast genomic DNA are cloned into T7 bacteriophage to create a translational fusion between the capsid protein and the peptide sequence encoded by the insert. (B) The library of phage are exposed to immobilized target DNA molecules and non-binding phage are washed away. Bound phage are eluted, amplified in liquid culture, and the process is repeated over multiple rounds. The sequence content of the enriched phage population is determined by PCR amplification of the inserts, labeling, and hybridization to a yeast ORF microarray.
    Figure Legend Snippet: An overview of Microarray profiling of phage-display selection technology. (A) 1–3 kb fragments of yeast genomic DNA are cloned into T7 bacteriophage to create a translational fusion between the capsid protein and the peptide sequence encoded by the insert. (B) The library of phage are exposed to immobilized target DNA molecules and non-binding phage are washed away. Bound phage are eluted, amplified in liquid culture, and the process is repeated over multiple rounds. The sequence content of the enriched phage population is determined by PCR amplification of the inserts, labeling, and hybridization to a yeast ORF microarray.

    Techniques Used: Microarray, Selection, Clone Assay, Sequencing, Binding Assay, Amplification, Polymerase Chain Reaction, Labeling, Hybridization

    The Rap1 DNA-binding domain is enriched in a sequence-specific and salt-dependent manner. A phage display library was affinity selected against indicated quantities of double-stranded DNA containing Rap1 binding sites under indicated salt conditions. Results from PCR of input phage library (input) or after second round of selection are shown. The intensity of a band is proportional to its abundance in the library. Lanes designated RAP1 indicate results of specific PCR against a single phage with the RAP1 DNA-binding domain known to be in the library and the red arrows mark the expected size of this clone. Gel-isolation and sequencing of the selected bands at this location confirmed that they correspond to this clone. The blue arrows point out the PCR products corresponding to the MCM1 DNA-binding domain (lower band) and MCM1 ORF (upper band). The remaining bands show variable enrichment as a function of salt concentration and likely represent non-specific enrichment during the selection.
    Figure Legend Snippet: The Rap1 DNA-binding domain is enriched in a sequence-specific and salt-dependent manner. A phage display library was affinity selected against indicated quantities of double-stranded DNA containing Rap1 binding sites under indicated salt conditions. Results from PCR of input phage library (input) or after second round of selection are shown. The intensity of a band is proportional to its abundance in the library. Lanes designated RAP1 indicate results of specific PCR against a single phage with the RAP1 DNA-binding domain known to be in the library and the red arrows mark the expected size of this clone. Gel-isolation and sequencing of the selected bands at this location confirmed that they correspond to this clone. The blue arrows point out the PCR products corresponding to the MCM1 DNA-binding domain (lower band) and MCM1 ORF (upper band). The remaining bands show variable enrichment as a function of salt concentration and likely represent non-specific enrichment during the selection.

    Techniques Used: Binding Assay, Sequencing, Polymerase Chain Reaction, Selection, Isolation, Concentration Assay

    MaPS identifies RAP1 as the gene whose product interacts with the Rap1 binding site. The yeast genomic DNA phage display library was selected for three rounds against a double-stranded oligonucleotide and PCR products of an upstream region containing Rap1 binding sites. The selected population of phage were profiled through microarray hybridization. Displayed is the distribution of the mean percentile rank for five independent such selections performed. The ORF corresponding to Rap1 had the highest mean percentile rank out of a total of 6242 ORFs queried on the array.
    Figure Legend Snippet: MaPS identifies RAP1 as the gene whose product interacts with the Rap1 binding site. The yeast genomic DNA phage display library was selected for three rounds against a double-stranded oligonucleotide and PCR products of an upstream region containing Rap1 binding sites. The selected population of phage were profiled through microarray hybridization. Displayed is the distribution of the mean percentile rank for five independent such selections performed. The ORF corresponding to Rap1 had the highest mean percentile rank out of a total of 6242 ORFs queried on the array.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Microarray, Hybridization

    10) Product Images from "Mapping of the juxtacentromeric heterochromatin-euchromatin frontier of human chromosome 21"

    Article Title: Mapping of the juxtacentromeric heterochromatin-euchromatin frontier of human chromosome 21

    Journal: Genome Research

    doi: 10.1101/gr.5440306

    ( A ) Bands of RT-PCR products in agarose gels after electrophoresis and staining with ethidium bromide. Four different cell lines were incubated with the drugs indicated above each panel. A testis cDNA library served as positive control. Genes are indicated
    Figure Legend Snippet: ( A ) Bands of RT-PCR products in agarose gels after electrophoresis and staining with ethidium bromide. Four different cell lines were incubated with the drugs indicated above each panel. A testis cDNA library served as positive control. Genes are indicated

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Staining, Incubation, cDNA Library Assay, Positive Control

    11) Product Images from "New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels"

    Article Title: New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004556

    MORF2-REP profiles of Ethiopian T . evansi stocks and T . evansi and T . brucei reference strains. 1.5% agarose gel showing MORF2-REP minisatellite PCR amplicons. Lane M: 100 bp plus marker, lanes 1 to 14: Ethiopian T . evansi stocks MCAM/ET/2013/MU/01-02-04-05-06-07-08-09-10-11-13-14-15-17, lane 15: T . b . gambiense LiTat 1.3, lane 16: T . b . brucei AnTat 1.1 E lane 17: T . evansi type A (RoTat 1.2), lane 18: T . evansi type B (KETRI 2479), lane 19: T . equiperdum Dodola 940, lane 20: T . b . gambiense ABBA, lane N: negative control
    Figure Legend Snippet: MORF2-REP profiles of Ethiopian T . evansi stocks and T . evansi and T . brucei reference strains. 1.5% agarose gel showing MORF2-REP minisatellite PCR amplicons. Lane M: 100 bp plus marker, lanes 1 to 14: Ethiopian T . evansi stocks MCAM/ET/2013/MU/01-02-04-05-06-07-08-09-10-11-13-14-15-17, lane 15: T . b . gambiense LiTat 1.3, lane 16: T . b . brucei AnTat 1.1 E lane 17: T . evansi type A (RoTat 1.2), lane 18: T . evansi type B (KETRI 2479), lane 19: T . equiperdum Dodola 940, lane 20: T . b . gambiense ABBA, lane N: negative control

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control

    12) Product Images from "Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis"

    Article Title: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001021

    Chromatin immunoprecipitation (ChIP) reveals co-occupancy at the promoters of Cic target genes by Atxn1 and Cic. (A) ChIP using Cic antisera confirmed Cic binding at the promoter of two direct targets of Capicua, Ccnd1 and Etv5 , that were up-regulated in the Atxn1 −/− and Atxn1 154Q/+ cerebella. (B) ChIP using Atxn1 anti-sera in cerebellar extracts from Atxn1 +/− , Atxn1 154Q/− , and Atxn1 −/− mice reveals a signal in mice expressing only wild-type ( Atxn1 +/− ) but not polyQ-Atxn1 ( Atxn1 154Q/− ) compared to negative controls (pre-immune sera, and Atxn1 −/− ) (C) ChIP as in (B), this time using Cic antibody. In contrast to (B), Cic is present at comparable levels at the target promoters in Atxn1 +/− , Atxn1 154Q/− , and Atxn −/− mice. All ChIP assays were repeated three times on independent samples, representative results shown. (D) ChIP followed by quantitative PCR (ChIP-qPCR) on the promoter of Etv5 confirms that the proportion of immunoprecipitated DNA by Cic antibody is comparable in all three genotypes (as seen in C). A region containing two Capicua binding sites (promoter-CBS) of Etv5 is more enriched by Cic antibody than a region lacking CBSs (promoter-no CBS) compared to preimmune sera. (E) ChIP-qPCR on the promoter of Ccnd1 also shows similar enrichment of immunoprecipitated DNA by Cic antibody in all three genotypes (as seen in C). ChIP-qPCR using primers designed for a region within 100 bps of the CBS in the Ccnd1 promoter (which is highly conserved across species) show more Cic binding than primers designed for a poorly conserved region further downstream (∼400 bps) of the CBS at the promoter of Ccnd1 , compared to preimmune sera. ChIP-qPCR experiments in (D) and (E) were performed in triplicate on three independent sets of samples (3 cerebella per genotype) using SYBR Green Dye. N.S. = not significant, ** p
    Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) reveals co-occupancy at the promoters of Cic target genes by Atxn1 and Cic. (A) ChIP using Cic antisera confirmed Cic binding at the promoter of two direct targets of Capicua, Ccnd1 and Etv5 , that were up-regulated in the Atxn1 −/− and Atxn1 154Q/+ cerebella. (B) ChIP using Atxn1 anti-sera in cerebellar extracts from Atxn1 +/− , Atxn1 154Q/− , and Atxn1 −/− mice reveals a signal in mice expressing only wild-type ( Atxn1 +/− ) but not polyQ-Atxn1 ( Atxn1 154Q/− ) compared to negative controls (pre-immune sera, and Atxn1 −/− ) (C) ChIP as in (B), this time using Cic antibody. In contrast to (B), Cic is present at comparable levels at the target promoters in Atxn1 +/− , Atxn1 154Q/− , and Atxn −/− mice. All ChIP assays were repeated three times on independent samples, representative results shown. (D) ChIP followed by quantitative PCR (ChIP-qPCR) on the promoter of Etv5 confirms that the proportion of immunoprecipitated DNA by Cic antibody is comparable in all three genotypes (as seen in C). A region containing two Capicua binding sites (promoter-CBS) of Etv5 is more enriched by Cic antibody than a region lacking CBSs (promoter-no CBS) compared to preimmune sera. (E) ChIP-qPCR on the promoter of Ccnd1 also shows similar enrichment of immunoprecipitated DNA by Cic antibody in all three genotypes (as seen in C). ChIP-qPCR using primers designed for a region within 100 bps of the CBS in the Ccnd1 promoter (which is highly conserved across species) show more Cic binding than primers designed for a poorly conserved region further downstream (∼400 bps) of the CBS at the promoter of Ccnd1 , compared to preimmune sera. ChIP-qPCR experiments in (D) and (E) were performed in triplicate on three independent sets of samples (3 cerebella per genotype) using SYBR Green Dye. N.S. = not significant, ** p

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, SYBR Green Assay

    13) Product Images from "Fusobacterium spp. target human CEACAM1 via the trimeric autotransporter adhesin CbpF"

    Article Title: Fusobacterium spp. target human CEACAM1 via the trimeric autotransporter adhesin CbpF

    Journal: Journal of Oral Microbiology

    doi: 10.1080/20002297.2018.1565043

    Comparison of CbpF presence in Fusobacterium species. (a) Western blots of lysates of Fusobacterium species nucleatum (Fn lane 1), vincentii (Fv lane 2), polymorphum (Fp lane 3), and a second vincentii strain (formerly F. nucleatum subspecies fusiforme (Fv2 lane 4)) were overlaid with anti-KW15, control pre-bleed serum, CEACAM1-Fc, and CD33-Fc as indicated. Note the CEACAM1-Fc and anti-KW15 binding bands in Fn, Fv, and Fv2 (*) but not in Fp. (b) PCR of cbpF from F . spp. Note the ~1,500 bp product for Fn, Fv, and Fv2 but no product as expected for Fp or Fa. Data are representative of two independent experiments.
    Figure Legend Snippet: Comparison of CbpF presence in Fusobacterium species. (a) Western blots of lysates of Fusobacterium species nucleatum (Fn lane 1), vincentii (Fv lane 2), polymorphum (Fp lane 3), and a second vincentii strain (formerly F. nucleatum subspecies fusiforme (Fv2 lane 4)) were overlaid with anti-KW15, control pre-bleed serum, CEACAM1-Fc, and CD33-Fc as indicated. Note the CEACAM1-Fc and anti-KW15 binding bands in Fn, Fv, and Fv2 (*) but not in Fp. (b) PCR of cbpF from F . spp. Note the ~1,500 bp product for Fn, Fv, and Fv2 but no product as expected for Fp or Fa. Data are representative of two independent experiments.

    Techniques Used: Western Blot, Binding Assay, Polymerase Chain Reaction

    Analyses of CEACAM1 binding and cbpF gene presence in clinical isolates of F. nucleatum . (a) Western blot of representative clinical Fn isolates overlaid with CEACAM1-Fc. Strain designations are indicated above each lane. Note the absence of binding for 2B6 compared to the other isolates which expressed a ~150 kDa CEACAM1-binding protein. (b) PCR for cbpF from representative clinical isolates. Strain designations are indicated above each lane. Note the lack of product for isolate 2B6 corresponding with a lack of CEACAM1 binding observed in A. (c) Analysis of CbpF migration from the two distinct phylogenetic groups. Western blot overlay indicating the relative migration of CbpF from group I (lane 1 and 4) and group II (lane 2). Lane 1, 2B17, lane 2 2B16, lane 3 2B14 (non-binding control), and lane 4 2B13. Blot was overlaid with CEACAM1-Fc at 1 μg ml −1 and detected via an alkaline phosphatase-conjugated secondary antibody. Data are representative of two independent experiments.
    Figure Legend Snippet: Analyses of CEACAM1 binding and cbpF gene presence in clinical isolates of F. nucleatum . (a) Western blot of representative clinical Fn isolates overlaid with CEACAM1-Fc. Strain designations are indicated above each lane. Note the absence of binding for 2B6 compared to the other isolates which expressed a ~150 kDa CEACAM1-binding protein. (b) PCR for cbpF from representative clinical isolates. Strain designations are indicated above each lane. Note the lack of product for isolate 2B6 corresponding with a lack of CEACAM1 binding observed in A. (c) Analysis of CbpF migration from the two distinct phylogenetic groups. Western blot overlay indicating the relative migration of CbpF from group I (lane 1 and 4) and group II (lane 2). Lane 1, 2B17, lane 2 2B16, lane 3 2B14 (non-binding control), and lane 4 2B13. Blot was overlaid with CEACAM1-Fc at 1 μg ml −1 and detected via an alkaline phosphatase-conjugated secondary antibody. Data are representative of two independent experiments.

    Techniques Used: Binding Assay, Western Blot, Polymerase Chain Reaction, Migration

    14) Product Images from "Infection with novel Bacteroides phage BV01 alters host transcriptome and bile acid metabolism in a common human gut microbe"

    Article Title: Infection with novel Bacteroides phage BV01 alters host transcriptome and bile acid metabolism in a common human gut microbe

    Journal: bioRxiv

    doi: 10.1101/2020.04.06.028910

    Prophage BV01 is an intact prophage. (A) Excision and circularization activities of the BV01 integrase are confirmed by PCR. The presence of phage DNA was detected by amplification of a phage marker gene (BVU_RS14350). Amplification across the phage attachment site ( attP ) indicates circularization of the BV01 genome; attP amplicons from the integrase complement strain (Δ int + pNBU2- int ) are ∼1.2 Kb shorter than wild-type amplicons due to deletion of the integrase gene. Supernatant fractions were treated with DNase, eliminating all contaminating host genomic DNA, as demonstrated by the amplification of a host marker gene (16S rRNA). Note that despite apparent size shift of BVU_RS14350 amplicons from the pellets and supernatants, Sanger sequencing validated that the products are in fact identical. PCR amplicons were visualized by agarose gel electrophoresis alongside NEB 1 Kb DNA ladder (BVU_RS14350, attP ) or GeneRuler Express DNA ladder (16S rRNA); ladder band sizes shown in Kb. (B) BV01 can transduce uninfected hosts in a gnotobiotic mouse. Germ-free mice ( n =7) were gavaged with an equal mixture of a BV01- tetQ lysogen and an erythromycin-tagged cured lysogen (Day 0). Recipient, donor, and transductant cells were identified by plating on Brain Heart Infusion (BHI) media with antibiotic selection: erythromycin (Erm) or tetracycline (Tet).
    Figure Legend Snippet: Prophage BV01 is an intact prophage. (A) Excision and circularization activities of the BV01 integrase are confirmed by PCR. The presence of phage DNA was detected by amplification of a phage marker gene (BVU_RS14350). Amplification across the phage attachment site ( attP ) indicates circularization of the BV01 genome; attP amplicons from the integrase complement strain (Δ int + pNBU2- int ) are ∼1.2 Kb shorter than wild-type amplicons due to deletion of the integrase gene. Supernatant fractions were treated with DNase, eliminating all contaminating host genomic DNA, as demonstrated by the amplification of a host marker gene (16S rRNA). Note that despite apparent size shift of BVU_RS14350 amplicons from the pellets and supernatants, Sanger sequencing validated that the products are in fact identical. PCR amplicons were visualized by agarose gel electrophoresis alongside NEB 1 Kb DNA ladder (BVU_RS14350, attP ) or GeneRuler Express DNA ladder (16S rRNA); ladder band sizes shown in Kb. (B) BV01 can transduce uninfected hosts in a gnotobiotic mouse. Germ-free mice ( n =7) were gavaged with an equal mixture of a BV01- tetQ lysogen and an erythromycin-tagged cured lysogen (Day 0). Recipient, donor, and transductant cells were identified by plating on Brain Heart Infusion (BHI) media with antibiotic selection: erythromycin (Erm) or tetracycline (Tet).

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Sequencing, Agarose Gel Electrophoresis, Mouse Assay, Selection

    15) Product Images from "Mutant Forkhead L2 (FOXL2) Proteins Associated with Premature Ovarian Failure (POF) Dimerize with Wild-Type FOXL2, Leading to Altered Regulation of Genes Associated with Granulosa Cell Differentiation"

    Article Title: Mutant Forkhead L2 (FOXL2) Proteins Associated with Premature Ovarian Failure (POF) Dimerize with Wild-Type FOXL2, Leading to Altered Regulation of Genes Associated with Granulosa Cell Differentiation

    Journal: Endocrinology

    doi: 10.1210/en.2010-0989

    ChIP suggests binding of FOXL2 to the human CYP19 promoter. KGN cells were transiently transfected with empty expression construct (control) or expression constructs for FLAG-tagged wild-type or mutant FOXL2. FLAG-tagged wild-type and mutant FOXL2 were then immunoprecipitated using the anti-FLAG M2 antibody. A, The amount of DNA cross-linked with FLAG-tagged wild-type or mutant FOXL2 was then measured by quantitative real-time PCR with primers corresponding to the −113- to +8-bp region of the human ovarian-specific CYP19 promoter. The results shown represent the averages of three independent experiments. The Ct counts for IP with IgG and M2 FLAG antibodies were first normalized against the Ct counts for 1% input, to give the percentage of total input. The percentages of total input from IP using the M2 FLAG antibody were then divided by the percentages of total input from IP using the IgG antibody. The results from ChIP with cells transfected with wild-type FOXL2 and mutant FOXL2 were further normalized against the results from ChIP with cells transfected with empty pFLAG-CMV-2 vector (control). Significant differences ( P
    Figure Legend Snippet: ChIP suggests binding of FOXL2 to the human CYP19 promoter. KGN cells were transiently transfected with empty expression construct (control) or expression constructs for FLAG-tagged wild-type or mutant FOXL2. FLAG-tagged wild-type and mutant FOXL2 were then immunoprecipitated using the anti-FLAG M2 antibody. A, The amount of DNA cross-linked with FLAG-tagged wild-type or mutant FOXL2 was then measured by quantitative real-time PCR with primers corresponding to the −113- to +8-bp region of the human ovarian-specific CYP19 promoter. The results shown represent the averages of three independent experiments. The Ct counts for IP with IgG and M2 FLAG antibodies were first normalized against the Ct counts for 1% input, to give the percentage of total input. The percentages of total input from IP using the M2 FLAG antibody were then divided by the percentages of total input from IP using the IgG antibody. The results from ChIP with cells transfected with wild-type FOXL2 and mutant FOXL2 were further normalized against the results from ChIP with cells transfected with empty pFLAG-CMV-2 vector (control). Significant differences ( P

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Transfection, Expressing, Construct, Mutagenesis, Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation

    16) Product Images from "Mutational processes shape the landscape of TP53 mutations in human cancer"

    Article Title: Mutational processes shape the landscape of TP53 mutations in human cancer

    Journal: Nature genetics

    doi: 10.1038/s41588-018-0204-y

    Comprehensive mutational scanning of TP53 ( a ) A library comprising 8258 mutant TP53 alleles was introduced into A549 p53 WT and p53 NULL cells in a pooled format under conditions that favor the integration of a single vector in each cell. Library-infected p53 WT cells were treated with nutlin-3, and library-infected p53 NULL cells were treated with either nutlin-3 or etoposide. After 12 days, genomic DNA was harvested, PCR-amplified and subjected to next generation sequencing. ( b–d ) Heat maps of normalized allele enrichment scores (Z-scores) with codon-level average Z-scores plotted at right. ( e ) Left , The reported domain structure of p53 with residues 175, 248, and 273 highlighted: TAD = transactivation domain, PRD = Proline-rich domain, DBD = DNA-binding domain, ZN = Zinc-binding domain, 4D = tetramerization domain, CTD = C-terminal domain. Right , Total number of missense and nonsense mutations found at each codon in the IARC database 2 , 32 . ( f – h ) Density plot of alleles with silent mutations (wild-type alleles), nonsense mutations at codons 44–289 (loss-of-function alleles), missense mutations that are common in cancer, and SNV-generated missense mutations that have never been observed in cancer. Differences among all groups of alleles were significant in each condition (P
    Figure Legend Snippet: Comprehensive mutational scanning of TP53 ( a ) A library comprising 8258 mutant TP53 alleles was introduced into A549 p53 WT and p53 NULL cells in a pooled format under conditions that favor the integration of a single vector in each cell. Library-infected p53 WT cells were treated with nutlin-3, and library-infected p53 NULL cells were treated with either nutlin-3 or etoposide. After 12 days, genomic DNA was harvested, PCR-amplified and subjected to next generation sequencing. ( b–d ) Heat maps of normalized allele enrichment scores (Z-scores) with codon-level average Z-scores plotted at right. ( e ) Left , The reported domain structure of p53 with residues 175, 248, and 273 highlighted: TAD = transactivation domain, PRD = Proline-rich domain, DBD = DNA-binding domain, ZN = Zinc-binding domain, 4D = tetramerization domain, CTD = C-terminal domain. Right , Total number of missense and nonsense mutations found at each codon in the IARC database 2 , 32 . ( f – h ) Density plot of alleles with silent mutations (wild-type alleles), nonsense mutations at codons 44–289 (loss-of-function alleles), missense mutations that are common in cancer, and SNV-generated missense mutations that have never been observed in cancer. Differences among all groups of alleles were significant in each condition (P

    Techniques Used: Mutagenesis, Plasmid Preparation, Infection, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing, Binding Assay, Generated

    17) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    18) Product Images from "YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth"

    Article Title: YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

    Journal: JNCI Journal of the National Cancer Institute

    doi: 10.1093/jnci/djr512

    Chromatin immunoprecipitation of E2F1 target gene promoters using YB-1 antibody. Chromatin from MCF-7 cells was cross-linked to fix bound proteins to the DNA. Cells were lysed and the chromatin was incubated with a YB-1 antibody to immunoprecipitate promoters bound by YB-1. Polymerase chain reaction was then performed to amplify promoter fragments of known E2F1-regulated genes to determine whether they were bound by YB-1. As controls, MCF-7 cells were transfected with siYB-1 72 hours before they were harvested to deplete YB-1, or chromatin was precleared with an E2F1 antibody to remove E2F1-bound promoters before incubation with YB-1 antibody. Input = MCF-7 DNA before immunoprecipitation; Beads = protein G beads in the absence of DNA; IgG = chromatin immunoprecipitation with the IgG negative control antibody; YB-1 ChIP = promoters immunoprecipitated with the YB-1 antibody; YB-1 = YB-1 antibody only; siYB-1 = cells pretreated with siYB-1; E2F1 = cells preincubated with E2F1 antibody. Figure shows typical results obtained from at least three independent experiments.
    Figure Legend Snippet: Chromatin immunoprecipitation of E2F1 target gene promoters using YB-1 antibody. Chromatin from MCF-7 cells was cross-linked to fix bound proteins to the DNA. Cells were lysed and the chromatin was incubated with a YB-1 antibody to immunoprecipitate promoters bound by YB-1. Polymerase chain reaction was then performed to amplify promoter fragments of known E2F1-regulated genes to determine whether they were bound by YB-1. As controls, MCF-7 cells were transfected with siYB-1 72 hours before they were harvested to deplete YB-1, or chromatin was precleared with an E2F1 antibody to remove E2F1-bound promoters before incubation with YB-1 antibody. Input = MCF-7 DNA before immunoprecipitation; Beads = protein G beads in the absence of DNA; IgG = chromatin immunoprecipitation with the IgG negative control antibody; YB-1 ChIP = promoters immunoprecipitated with the YB-1 antibody; YB-1 = YB-1 antibody only; siYB-1 = cells pretreated with siYB-1; E2F1 = cells preincubated with E2F1 antibody. Figure shows typical results obtained from at least three independent experiments.

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Polymerase Chain Reaction, Transfection, Immunoprecipitation, Negative Control

    19) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    20) Product Images from "Identification of cis-Acting Nucleotides and a Structural Feature in West Nile Virus 3?-Terminus RNA That Facilitate Viral Minus Strand RNA Synthesis"

    Article Title: Identification of cis-Acting Nucleotides and a Structural Feature in West Nile Virus 3?-Terminus RNA That Facilitate Viral Minus Strand RNA Synthesis

    Journal: Journal of Virology

    doi: 10.1128/JVI.00212-13

    Analysis of the effect of WNV 3′ SL mutations on the translation and replication of viral RNA. (A) Translated WNV proteins (red) present in BHK cells 3 h after transfection of genomic viral RNA were detected by confocal microscopy using anti-WNV MHIAF. (B) Relative fluorescent intensities of WNV proteins in the cytoplasm of transfected BHK cells determined as described in Materials and Methods. (C) Relative fluorescence intensities of WNV protein in the cytoplasm of BHK cells transfected with either capped, uncapped, or different ratios (1:9 and 1:19) of capped to uncapped in vitro transcribe full-length viral RNAs. Error bars represent the SE ( n = 3). (D) Relative quantification of intracellular WNV minus-strand RNA levels by strand-specific real-time qRT-PCR. Minus strand RNA levels are expressed as a log 10 fold change compared to the level of RNA detected at 2 h after viral RNA transfection. Each RNA sample was normalized to cellular GAPDH mRNA in the same sample. Error bars represent the standard error (SE). Each sample was assayed in triplicate and the data shown are representative of three independent experiments. (E) As an alternative means of quantifying minus strand viral RNA, minus strand viral RNA present in extracted total cell RNA was hybridized to a biotin-labeled complementary probe and purified using streptavidin magnetic beads, and the amount of minus viral RNA was measured by absolute real-time qRT-PCR. (F) Relative quantification of intracellular WNV genomic (positive-strand) RNA levels by real-time qRT-PCR. Genomic RNA levels are expressed as a log fold change of RNA compared to the level of RNA present 6 h after transfection. Each RNA sample was normalized to cellular GAPDH mRNA in the same sample. Error bars represent SE ( n = 3). Each sample was assayed in triplicate, and the data shown are representative of three independent experiments.
    Figure Legend Snippet: Analysis of the effect of WNV 3′ SL mutations on the translation and replication of viral RNA. (A) Translated WNV proteins (red) present in BHK cells 3 h after transfection of genomic viral RNA were detected by confocal microscopy using anti-WNV MHIAF. (B) Relative fluorescent intensities of WNV proteins in the cytoplasm of transfected BHK cells determined as described in Materials and Methods. (C) Relative fluorescence intensities of WNV protein in the cytoplasm of BHK cells transfected with either capped, uncapped, or different ratios (1:9 and 1:19) of capped to uncapped in vitro transcribe full-length viral RNAs. Error bars represent the SE ( n = 3). (D) Relative quantification of intracellular WNV minus-strand RNA levels by strand-specific real-time qRT-PCR. Minus strand RNA levels are expressed as a log 10 fold change compared to the level of RNA detected at 2 h after viral RNA transfection. Each RNA sample was normalized to cellular GAPDH mRNA in the same sample. Error bars represent the standard error (SE). Each sample was assayed in triplicate and the data shown are representative of three independent experiments. (E) As an alternative means of quantifying minus strand viral RNA, minus strand viral RNA present in extracted total cell RNA was hybridized to a biotin-labeled complementary probe and purified using streptavidin magnetic beads, and the amount of minus viral RNA was measured by absolute real-time qRT-PCR. (F) Relative quantification of intracellular WNV genomic (positive-strand) RNA levels by real-time qRT-PCR. Genomic RNA levels are expressed as a log fold change of RNA compared to the level of RNA present 6 h after transfection. Each RNA sample was normalized to cellular GAPDH mRNA in the same sample. Error bars represent SE ( n = 3). Each sample was assayed in triplicate, and the data shown are representative of three independent experiments.

    Techniques Used: Transfection, Confocal Microscopy, Fluorescence, In Vitro, Quantitative RT-PCR, Labeling, Purification, Magnetic Beads

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    Article Snippet: .. The reactions were pooled and purified (Qiagen MinElute PCR cleanup kit). .. High throughput sequencing and analysis The amplified cDNA was utilized in high-throughput nucleic acid sequencing using Genome Sequencer FLX Titanium pyrosequencing technology and reagents (Roche).

    Modification:

    Article Title: Copy Number Variation Is a Fundamental Aspect of the Placental Genome
    Article Snippet: .. Libraries for WGS were prepared from 40–50 ng genomic DNA using the Nextera TruSeq Dual Index Paired End Kit (Illumina) following manufacturer's instructions with the following modification: the Qiagen MinElute Reaction Cleanup Kit (Qiagen) was used to cleanup Tagmented DNA. .. Library quality was assessed using Qubit and Bioanalyzer, and sequenced on the Illumina HiSeq 2000 at approximately 10× coverage ( ) at the Stanford Center for Genomics and Personalized Medicine.

    Incubation:

    Article Title: Reverse gene-environment interaction approach to identify variants influencing body-mass index in humans
    Article Snippet: .. We incubated at 37ºC for 30 minutes, and then purified the DNA with the Qiagen MinElute kit (Qiagen 28204). .. Libraries were amplified using 6 cycles and sequenced on the Illumina HiSeq 4000 to produce an average of 23,376,290 (+/− 3,337,206) reads.

    Amplification:

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    Purification:

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    Article Snippet: .. Half of the fragmented DNA (50 μL) was purified using a MinElute Reaction Cleanup kit (Qiagen) and eluted in 25 μL EB buffer. .. End repair and A-tailing was done on the purified DNA (25 μL) using DNA T4 Polymerase (7.5 U), T4 Polynucleotide Kinase (25 U), dNTP (0.2 mM), DreamTaq DNA Polymerase (1.25 U), ATP (5 mM), 2.5 μL T4 Polynucleotide Kinase Buffer A (10x) and 5 μL T4 DNA Polymerase Buffer, in a total volume of 50 μL (all enzymes from Fermentas).

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    FOXC1 binds to the Msx2 promoter in vivo. (A) Sequence analysis of upstream regulatory elements reveals the presence of a FOXC1 binding motif, indicated in bold, (TAAAT/CAAT) located in a conserved motif near the predicted Msx2 transcription start site of the mouse, rat and human genes. Small arrows correspond to the position of ChIP primers located in the promoter region or the coding region of mouse Msx2 . Nucleotide sequences of the Electrophoretic mobility shift assay (EMSA) probes for wild type (WT) and mutated (MUT) FOXC1 binding sites are indicated. (B) Chromatin immunoprecipitation assays confirm the binding of FOXC1 to the Msx2 promoter in vivo . Quantitative <t>PCR</t> (qPCR) was conducted on ChiP products isolated from 10T1/2 cells using antibodies recognizing FOXC1 or normal immunoglobulins (IgG). Primers were designed amplify regions in the promoter flanking the putative FOXC1 binding site or exon 2 of the mouse Msx2 gene. Amplification signals are presented a percentage compared to input chromatin fraction. (C) EMSAs demonstrate FOXC1 binding to <t>DNA</t> elements in the Msx2 promoter. Extracts from U2OS cells or cells transfected with FOXC1 were incubated with IR700-labeled oligonucleotides correspond to the WT or MUT FOXC1 binding sites. FOXC1-DNA complexes are indicated by the arrow.
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    FOXC1 binds to the Msx2 promoter in vivo. (A) Sequence analysis of upstream regulatory elements reveals the presence of a FOXC1 binding motif, indicated in bold, (TAAAT/CAAT) located in a conserved motif near the predicted Msx2 transcription start site of the mouse, rat and human genes. Small arrows correspond to the position of ChIP primers located in the promoter region or the coding region of mouse Msx2 . Nucleotide sequences of the Electrophoretic mobility shift assay (EMSA) probes for wild type (WT) and mutated (MUT) FOXC1 binding sites are indicated. (B) Chromatin immunoprecipitation assays confirm the binding of FOXC1 to the Msx2 promoter in vivo . Quantitative PCR (qPCR) was conducted on ChiP products isolated from 10T1/2 cells using antibodies recognizing FOXC1 or normal immunoglobulins (IgG). Primers were designed amplify regions in the promoter flanking the putative FOXC1 binding site or exon 2 of the mouse Msx2 gene. Amplification signals are presented a percentage compared to input chromatin fraction. (C) EMSAs demonstrate FOXC1 binding to DNA elements in the Msx2 promoter. Extracts from U2OS cells or cells transfected with FOXC1 were incubated with IR700-labeled oligonucleotides correspond to the WT or MUT FOXC1 binding sites. FOXC1-DNA complexes are indicated by the arrow.

    Journal: PLoS ONE

    Article Title: Initiation of Early Osteoblast Differentiation Events through the Direct Transcriptional Regulation of Msx2 by FOXC1

    doi: 10.1371/journal.pone.0049095

    Figure Lengend Snippet: FOXC1 binds to the Msx2 promoter in vivo. (A) Sequence analysis of upstream regulatory elements reveals the presence of a FOXC1 binding motif, indicated in bold, (TAAAT/CAAT) located in a conserved motif near the predicted Msx2 transcription start site of the mouse, rat and human genes. Small arrows correspond to the position of ChIP primers located in the promoter region or the coding region of mouse Msx2 . Nucleotide sequences of the Electrophoretic mobility shift assay (EMSA) probes for wild type (WT) and mutated (MUT) FOXC1 binding sites are indicated. (B) Chromatin immunoprecipitation assays confirm the binding of FOXC1 to the Msx2 promoter in vivo . Quantitative PCR (qPCR) was conducted on ChiP products isolated from 10T1/2 cells using antibodies recognizing FOXC1 or normal immunoglobulins (IgG). Primers were designed amplify regions in the promoter flanking the putative FOXC1 binding site or exon 2 of the mouse Msx2 gene. Amplification signals are presented a percentage compared to input chromatin fraction. (C) EMSAs demonstrate FOXC1 binding to DNA elements in the Msx2 promoter. Extracts from U2OS cells or cells transfected with FOXC1 were incubated with IR700-labeled oligonucleotides correspond to the WT or MUT FOXC1 binding sites. FOXC1-DNA complexes are indicated by the arrow.

    Article Snippet: Qiagen PCR cleanup kit was used to isolate ChIP DNA.

    Techniques: In Vivo, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Electrophoretic Mobility Shift Assay, Real-time Polymerase Chain Reaction, Isolation, Amplification, Transfection, Incubation, Labeling

    Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative RT-PCR analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic DNA samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.

    Journal: Genes & Development

    Article Title: Chromatin restriction by the nucleosome remodeler Mi-2β and functional interplay with lineage-specific transcription regulators control B-cell differentiation

    doi: 10.1101/gad.321901.118

    Figure Lengend Snippet: Igh rearrangement is not dependent on Mi-2β. ( A ) Diagram of Igh locus depicting proximal and distal V , D , and J clusters tested for recombination. The five V H families are represented by white boxes, and the D , J , and C regions are shown by black boxes. Rearrangement of the most distal variable gene ( V H -558 ) and the two most proximal ( V H -Q52 and V H -7183 ) was analyzed. The initiation sites for the μ0 and Iμ germline transcripts are depicted. ( B ) Semiquantitative RT-PCR analyses for Igh germline transcripts, Chd4 , and its homolog, Chd3 , in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and samples were normalized using Gapdh expression. ( C ) VDJ rearrangement in WT and ΔChd4 Cd2 pro-B cells. ( Top ) Normalized genomic DNA samples from sorted pro-B populations were analyzed for rearrangement by PCR amplification followed by Southern blotting. Fivefold dilutions of gDNA were used for PCR amplification. Rearrangement of D-J as well as V H 558 , V H -Q52 , and V H -7183 to DJ , respectively, is shown for both WT and ΔChd4 Cd2 . ( Bottom ) The deletion of the ATPase subunit of Mi-2β ( CHD4 ) was verified by PCR amplification of exons 11–23. The samples were normalized to an amplified Ikzf1 fragment. ( D ) Flow cytometry for expression of intracellular IgM ( ICμ ) in pro-B and small pre-B cells from WT and ΔChd4 Cd2 mice. Numbers indicate the percentage of cells in the ICμ -high gate. ( E ) Semiquantitative RT-PCR analyses for early B-cell differentiation markers ( Flt3 , Il7r , Ebf1 , and Pax5 ), key regulators for Ig recombination, and pre-BCR complex components ( Rag1 , Rag2 , Dntt , Vpreb1 , and Igll1 ) in pro-B cells from WT and ΔChd4 Cd2 mice. Fivefold dilutions of cDNA were used, and the samples were normalized using Gapdh expression.

    Article Snippet: Amplified DNA was purified with a Qiagen PCR cleanup kit.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Expressing, Polymerase Chain Reaction, Amplification, Southern Blot, Flow Cytometry, Cytometry, Cell Differentiation

    ETO interacts directly with C/EBPβ inhibiting its DNA-binding activity toward the C/EBPα promoter and preventing centromeric localization during adipogenesis. (A) HEK293 cells were transfected with control vector, GFP-ETO (G-ETO-WT), or GFP-ETO-AA in the absence or presence of C/EBPβ as indicated. Anti-C/EBPβ immunoprecipitates were analyzed for associated ETO protein (upper panel), whereas corresponding cell lysates were probed for ETO (middle panel) or C/EBPβ (lower panel) by Western blotting. (B) 3T3-L1 preadipocytes expressing GFP-ETO were treated for 4 h in the absence or presence of differentiation cocktail as indicated prior to lysis. Cell lysates (left panels) or immunoprecipitates prepared by using an anti-ETO antibody (Santa Cruz Biotechnology) (right panels) were analyzed by Western blotting to detect ETO (upper panels) or C/EBPβ isoforms (lower panels). (C) In vitro transcribed/translated C/EBPβ (Cβ) and/or ETO (E) were incubated with radiolabeled DNA probe corresponding to the proximal C/EBPα binding site of the C/EBPα promoter in a gel shift assay. Various ratios of C/EBPβ to ETO were achieved by adjusting the quantity of ETO. In all lanes total protein input was kept constant by appropriate addition of rabbit reticulocyte lysate, except in lane 1, where free labeled probe was run alone. (D) 3T3-L1 preadipocytes were induced to differentiate for the times indicated and ChIP assays performed by using an anti-C/EBPβ antibody to isolate C/EBPβ-associated DNA. DNA from these immunoprecipitates corresponding to the C/EBPβ binding site in the C/EBPα promoter was quantified by using real-time PCR and normalized to DNA from a 10% sample of corresponding input lysate. The data are means ± the SEM from three independent experiments. Asterisks indicate a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: ETO/MTG8 Is an Inhibitor of C/EBP? Activity and a Regulator of Early Adipogenesis

    doi: 10.1128/MCB.24.22.9863-9872.2004

    Figure Lengend Snippet: ETO interacts directly with C/EBPβ inhibiting its DNA-binding activity toward the C/EBPα promoter and preventing centromeric localization during adipogenesis. (A) HEK293 cells were transfected with control vector, GFP-ETO (G-ETO-WT), or GFP-ETO-AA in the absence or presence of C/EBPβ as indicated. Anti-C/EBPβ immunoprecipitates were analyzed for associated ETO protein (upper panel), whereas corresponding cell lysates were probed for ETO (middle panel) or C/EBPβ (lower panel) by Western blotting. (B) 3T3-L1 preadipocytes expressing GFP-ETO were treated for 4 h in the absence or presence of differentiation cocktail as indicated prior to lysis. Cell lysates (left panels) or immunoprecipitates prepared by using an anti-ETO antibody (Santa Cruz Biotechnology) (right panels) were analyzed by Western blotting to detect ETO (upper panels) or C/EBPβ isoforms (lower panels). (C) In vitro transcribed/translated C/EBPβ (Cβ) and/or ETO (E) were incubated with radiolabeled DNA probe corresponding to the proximal C/EBPα binding site of the C/EBPα promoter in a gel shift assay. Various ratios of C/EBPβ to ETO were achieved by adjusting the quantity of ETO. In all lanes total protein input was kept constant by appropriate addition of rabbit reticulocyte lysate, except in lane 1, where free labeled probe was run alone. (D) 3T3-L1 preadipocytes were induced to differentiate for the times indicated and ChIP assays performed by using an anti-C/EBPβ antibody to isolate C/EBPβ-associated DNA. DNA from these immunoprecipitates corresponding to the C/EBPβ binding site in the C/EBPα promoter was quantified by using real-time PCR and normalized to DNA from a 10% sample of corresponding input lysate. The data are means ± the SEM from three independent experiments. Asterisks indicate a statistically significant difference ( P

    Article Snippet: Samples were incubated at 65°C for 6 h, and DNA was isolated by using a Qiagen PCR cleanup kit according to the manufacturer's instructions.

    Techniques: Binding Assay, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Lysis, In Vitro, Incubation, Electrophoretic Mobility Shift Assay, Labeling, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction