pcr product purification kits  (Qiagen)

 
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    Name:
    MinElute PCR Purification Kit
    Description:
    For purification of up to 5 μg PCR products 70 bp to 4 kb in low elution volumes Kit contents Qiagen MinElute PCR Purification Kit 50 MinElute Spin Columns 5g Binding Capacity 10L Elution Volume Tube Format Silica Technology 70 bp to 4 kb Fragment Size Manual Processing DNA Sample Fast Procedure and Easy Handling High Reproducible Recoveries For Purification of up to 5μg PCR Products in Low Elution Volumes Includes 50 MinElute Spin Columns Buffers 2mL Collection Tubes Benefits Very small elution volumes Fast procedure and easy handling High reproducible recoveries Gel loading dye for convenient sample analysis
    Catalog Number:
    28004
    Price:
    134
    Category:
    MinElute PCR Purification Kit
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    Structured Review

    Qiagen pcr product purification kits
    MinElute PCR Purification Kit
    For purification of up to 5 μg PCR products 70 bp to 4 kb in low elution volumes Kit contents Qiagen MinElute PCR Purification Kit 50 MinElute Spin Columns 5g Binding Capacity 10L Elution Volume Tube Format Silica Technology 70 bp to 4 kb Fragment Size Manual Processing DNA Sample Fast Procedure and Easy Handling High Reproducible Recoveries For Purification of up to 5μg PCR Products in Low Elution Volumes Includes 50 MinElute Spin Columns Buffers 2mL Collection Tubes 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 product purification kits/product/Qiagen
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    pcr product purification kits - by Bioz Stars, 2021-02
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    Images

    1) Product Images from "Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase"

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115318

    Single fragment cloning by Hot Fusion. (A) A diagram of single fragment cloning in a binary base vector. Base vector containing a lacZ gene was linearized by restriction enzyme digestion with AscI and KpnI to release lacZ . The linearized vector was directly used for Hot Fusion. (B) Amplified PCR products (A1 through A2) of plant gene promoters were used for cloning. M is a NEB 1 kb DNA ladder. (C) Transformation plates of cloned PCR products (A1 and H1 are not shown). Blue colonies on the plates contain the parental vector and white colonies contain the potential recombinants. Eight white colonies were screened for each construct ( Table 2 ).
    Figure Legend Snippet: Single fragment cloning by Hot Fusion. (A) A diagram of single fragment cloning in a binary base vector. Base vector containing a lacZ gene was linearized by restriction enzyme digestion with AscI and KpnI to release lacZ . The linearized vector was directly used for Hot Fusion. (B) Amplified PCR products (A1 through A2) of plant gene promoters were used for cloning. M is a NEB 1 kb DNA ladder. (C) Transformation plates of cloned PCR products (A1 and H1 are not shown). Blue colonies on the plates contain the parental vector and white colonies contain the potential recombinants. Eight white colonies were screened for each construct ( Table 2 ).

    Techniques Used: Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Transformation Assay, Construct

    PCR screening of multi-fragment assembled clones from Table 3 . Eight clones (colonies) from each construct were grown overnight in the 96-well plate containing LB + Spectinomycin (50 ug/ml). One µl of the over-night grown cell culture was used for PCR screening with base vector primers (18 bp) located immediately upstream of the promoter and downstream of the terminator. PCR products for each construct were loaded every other lane on a 1.0% agarose gel (containing ethidium bromide) with a multi channel pipette. GeneRuler 1 kb Plus DNA Ladder was used as the DNA marker.
    Figure Legend Snippet: PCR screening of multi-fragment assembled clones from Table 3 . Eight clones (colonies) from each construct were grown overnight in the 96-well plate containing LB + Spectinomycin (50 ug/ml). One µl of the over-night grown cell culture was used for PCR screening with base vector primers (18 bp) located immediately upstream of the promoter and downstream of the terminator. PCR products for each construct were loaded every other lane on a 1.0% agarose gel (containing ethidium bromide) with a multi channel pipette. GeneRuler 1 kb Plus DNA Ladder was used as the DNA marker.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Construct, Cell Culture, Plasmid Preparation, Agarose Gel Electrophoresis, Transferring, Marker

    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
    Figure Legend Snippet: A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Generated, Transformation Assay

    2) Product Images from "Rapid one-step recombinational cloning"

    Article Title: Rapid one-step recombinational cloning

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn167

    PCR confirmation of genes cloned into Kan-resistant vector (top panel) and Spec-resistant vector (bottom panel). Both cultured cells and plasmid DNAs were used for PCR confirmation. The 1 kb DNA marker (New England Biolabs) was used between every16 lanes. Samples were loaded using a multi-channel pipette, which loads every other well resulting in an interleaved loading pattern. In the upper panel, four individual colonies were screened for each clone using universal primers, which anneal to the vector sequences. Accordingly, clones without the insert amplify a small band of 200 bp. In the bottom panel, eight colonies from each clone were screened using gene-specific primers, so no band is observed for clones that do not contain the desired insert. While the use of gene-specific primers is useful for confirming the identity of a given clone, false negatives can arise due to PCR failures.
    Figure Legend Snippet: PCR confirmation of genes cloned into Kan-resistant vector (top panel) and Spec-resistant vector (bottom panel). Both cultured cells and plasmid DNAs were used for PCR confirmation. The 1 kb DNA marker (New England Biolabs) was used between every16 lanes. Samples were loaded using a multi-channel pipette, which loads every other well resulting in an interleaved loading pattern. In the upper panel, four individual colonies were screened for each clone using universal primers, which anneal to the vector sequences. Accordingly, clones without the insert amplify a small band of 200 bp. In the bottom panel, eight colonies from each clone were screened using gene-specific primers, so no band is observed for clones that do not contain the desired insert. While the use of gene-specific primers is useful for confirming the identity of a given clone, false negatives can arise due to PCR failures.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Cell Culture, Marker, Transferring

    Comparison of the PCR results using truncated attL sites and different DNA polymerases. HiFi tag refers to high-fidelity tag polymerase. Hercu refers to herculase. Pfu refers to Pfu turbo polymerases.
    Figure Legend Snippet: Comparison of the PCR results using truncated attL sites and different DNA polymerases. HiFi tag refers to high-fidelity tag polymerase. Hercu refers to herculase. Pfu refers to Pfu turbo polymerases.

    Techniques Used: Polymerase Chain Reaction

    3) Product Images from "Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens"

    Article Title: Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.12.8634-8641.2005

    Expression of omcS , omcT , and omcE . (A) The expression of omcS , omcT , and omcE was measured by RT-PCR. RNAs were extracted from wild-type cells grown in the presence of fumarate (F), ferric citrate (Fe sol ), and insoluble Fe(III) oxide (Fe insol ). Negative (−) controls were performed without cDNA synthesis, and positive controls (+) were performed by using genomic DNA as the template. (B) Northern analysis of omcS and omcT expression in cells grown on fumarate. The expression of omcS in the wild type (lane 1) and the Δ omcS :: spec (lane 2) and Δ omcT :: spec (lane 3) mutants is shown. The expression of omcT in the wild type (lane 4) and the Δ omcS :: spec (lane 5) and Δ omcT :: spec (lane 6) mutants is also indicated. (C) Expression of omcS and omcT in deletion mutants. The strains were cultured with fumarate as the electron acceptor, and genomic DNA was used as a template for positive controls. MW, molecular size marker.
    Figure Legend Snippet: Expression of omcS , omcT , and omcE . (A) The expression of omcS , omcT , and omcE was measured by RT-PCR. RNAs were extracted from wild-type cells grown in the presence of fumarate (F), ferric citrate (Fe sol ), and insoluble Fe(III) oxide (Fe insol ). Negative (−) controls were performed without cDNA synthesis, and positive controls (+) were performed by using genomic DNA as the template. (B) Northern analysis of omcS and omcT expression in cells grown on fumarate. The expression of omcS in the wild type (lane 1) and the Δ omcS :: spec (lane 2) and Δ omcT :: spec (lane 3) mutants is shown. The expression of omcT in the wild type (lane 4) and the Δ omcS :: spec (lane 5) and Δ omcT :: spec (lane 6) mutants is also indicated. (C) Expression of omcS and omcT in deletion mutants. The strains were cultured with fumarate as the electron acceptor, and genomic DNA was used as a template for positive controls. MW, molecular size marker.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Cell Culture, Marker

    4) Product Images from "HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin"

    Article Title: HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2565

    Increased H3 turnover in heterochromatin mutants is not solely due to changes in RNAPII transcription ( a ) Histone H3 replacement (blue) was measured across the mating type locus using MNase–ChIP–on–Chip analysis as described in Fig 1 . RNAPII occupancy (ChIP vs Input) was measured by ChIP–on–Chip in clr3 Δ, clr4 Δ or wild–type (WT) cells and plotted in alignment with the map (red). ( b ) RT–PCR analysis performed using total RNA samples isolated from clr3 Δ, clr4 Δ or wild–type (WT) cells. Genomic DNA (gDNA) was used as a control. The locations amplified by primer pairs 49, 51, 65 and 70 are highlighted with red shading (see online methods for primer references). Heterochromatin and euchromatin portions of the mating–type region are indicated at the top.
    Figure Legend Snippet: Increased H3 turnover in heterochromatin mutants is not solely due to changes in RNAPII transcription ( a ) Histone H3 replacement (blue) was measured across the mating type locus using MNase–ChIP–on–Chip analysis as described in Fig 1 . RNAPII occupancy (ChIP vs Input) was measured by ChIP–on–Chip in clr3 Δ, clr4 Δ or wild–type (WT) cells and plotted in alignment with the map (red). ( b ) RT–PCR analysis performed using total RNA samples isolated from clr3 Δ, clr4 Δ or wild–type (WT) cells. Genomic DNA (gDNA) was used as a control. The locations amplified by primer pairs 49, 51, 65 and 70 are highlighted with red shading (see online methods for primer references). Heterochromatin and euchromatin portions of the mating–type region are indicated at the top.

    Techniques Used: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification

    Clr3–dependent suppression of histone turnover correlates with epigenetic stability of heterochromatin ( a ) ChIP analysis of Clr3 localization of at the silent mat region. Strains expressing Myc tagged Clr3 in K Δ ∷ura4 + ura4–on or ura4–off state were used to perform ChIP. ChIP DNA was analyzed by semi–quantitative competitive PCR using primers that amplify both full–length K Δ ∷ura4 + and endogenous mini– ura4 ( ura4DSE ) as internal control. The relative enrichments were determined by calculating the ratio of the band intensities of [ChIP K Δ ∷ura4 + ÷ ChIP ura4DSE ] ÷ [Input K Δ ∷ura4 + ÷ Input ura4DSE ]. Results were confirmed by quantitative real–time PCR (qPCR). Relative enrichment of K Δ ∷ura4 + was normalized against untagged negative control and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 3 independent biological replicates (n=3) ( b ) H3 replacement was measured in K Δ ∷ura4 + ura4–on or ura4–off cells. The endogenous ura4 + was deleted in the strains used. ( c ) ChIP analysis of H3K9me2 levels at K Δ ∷ura4 + ura4–on cells. Experiments were performed with the same strains used in a . H3K9me levels were confirmed by qPCR and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 4 independent biological replicates (n=4).
    Figure Legend Snippet: Clr3–dependent suppression of histone turnover correlates with epigenetic stability of heterochromatin ( a ) ChIP analysis of Clr3 localization of at the silent mat region. Strains expressing Myc tagged Clr3 in K Δ ∷ura4 + ura4–on or ura4–off state were used to perform ChIP. ChIP DNA was analyzed by semi–quantitative competitive PCR using primers that amplify both full–length K Δ ∷ura4 + and endogenous mini– ura4 ( ura4DSE ) as internal control. The relative enrichments were determined by calculating the ratio of the band intensities of [ChIP K Δ ∷ura4 + ÷ ChIP ura4DSE ] ÷ [Input K Δ ∷ura4 + ÷ Input ura4DSE ]. Results were confirmed by quantitative real–time PCR (qPCR). Relative enrichment of K Δ ∷ura4 + was normalized against untagged negative control and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 3 independent biological replicates (n=3) ( b ) H3 replacement was measured in K Δ ∷ura4 + ura4–on or ura4–off cells. The endogenous ura4 + was deleted in the strains used. ( c ) ChIP analysis of H3K9me2 levels at K Δ ∷ura4 + ura4–on cells. Experiments were performed with the same strains used in a . H3K9me levels were confirmed by qPCR and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 4 independent biological replicates (n=4).

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Negative Control

    5) Product Images from "Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression"

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression

    Journal:

    doi: 10.1182/blood-2006-11-053710

    Clonality of serially transplanted long-term hematopoiesis. Representative LAM-PCR analysis of peripheral blood. Multiple clones contributed to the long-term hematopoiesis of the primary (A), secondary (B), and tertiary recipient mouse (C), and the number
    Figure Legend Snippet: Clonality of serially transplanted long-term hematopoiesis. Representative LAM-PCR analysis of peripheral blood. Multiple clones contributed to the long-term hematopoiesis of the primary (A), secondary (B), and tertiary recipient mouse (C), and the number

    Techniques Used: Laser Capture Microdissection, Polymerase Chain Reaction, Clone Assay

    The number of clones contributing to marked hematopoiesis is not affected by BCNU/O6BG treatment. Representative LAM-PCR analysis of peripheral blood of mice at different time points after transplantation of 4 × 10 5 cells. Peripheral blood samples
    Figure Legend Snippet: The number of clones contributing to marked hematopoiesis is not affected by BCNU/O6BG treatment. Representative LAM-PCR analysis of peripheral blood of mice at different time points after transplantation of 4 × 10 5 cells. Peripheral blood samples

    Techniques Used: Clone Assay, Laser Capture Microdissection, Polymerase Chain Reaction, Mouse Assay, Transplantation Assay

    6) Product Images from "Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms"

    Article Title: Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky016

    Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.
    Figure Legend Snippet: Schematic of in situ replication of DNA molecules from next-generation sequencing (NGS) platforms and subsequent PCR-based retrieval of target sequences. ( A ) Process flow chart for PCR-based methods for the retrieval of error-free DNA targets from an NGS-replica pool. ( B ) Preparation strategy of 454 GS Junior sequencing-based retrieval. Combinatorial barcode-tagged (CBT) pools were processed from microarray-synthesized oligonucleotides and subsequently ligated to the sheared genomic DNA as flanking sequences. The library was replicated in a sealed NGS plate. ( C ) Preparation strategy of a pre-NGS pool (MiSeq and Ion Proton). The barcoded library (cgc50 pool) was directly synthesized on a microarray. ( D ) Schematic of library replication in a MiSeq flow cell. ( E ) Schematic of library replication using melt-off DNA in the Ion Proton system. This process could be automatically performed using an Ion OneTouch™ ES system.

    Techniques Used: In Situ, Next-Generation Sequencing, Polymerase Chain Reaction, Flow Cytometry, Sequencing, Microarray, Synthesized

    7) Product Images from "Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension"

    Article Title: Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension

    Journal: Nature communications

    doi: 10.1038/ncomms7252

    Screening animals for ZFN-targeted mutation at the Nr2f2 locus (A) Tail DNA samples from pups born post-microinjection of custom ZFNs targeting exon 2 of the Nr2f2 locus were screened by PCR amplification with primers designed to amplify rat genomic fragments encompassing the ZFN-targeted site. Deviations from the expected product size of 360bp were inferred as genomic DNA from rats with mutations at the Nr2f2 locus. (B) Representative sequencing results from the PCR products shown in panel A detected a 15 bp deletion in the mRNA from mutant rats. Also represented are the corresponding translated peptide sequences as a result of the nucleotide variations.
    Figure Legend Snippet: Screening animals for ZFN-targeted mutation at the Nr2f2 locus (A) Tail DNA samples from pups born post-microinjection of custom ZFNs targeting exon 2 of the Nr2f2 locus were screened by PCR amplification with primers designed to amplify rat genomic fragments encompassing the ZFN-targeted site. Deviations from the expected product size of 360bp were inferred as genomic DNA from rats with mutations at the Nr2f2 locus. (B) Representative sequencing results from the PCR products shown in panel A detected a 15 bp deletion in the mRNA from mutant rats. Also represented are the corresponding translated peptide sequences as a result of the nucleotide variations.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Sequencing

    8) Product Images from "Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68?C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation †"

    Article Title: Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68?C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.5.2893-2898.2003

    DGGE patterns of PCR-amplified 16S rDNA segments. Each lane represents a 100-μm vertical interval through the microbial mat. Bands 1 and 2 are cyanobacterial sequences; the bands labeled nc are noncyanobacterial sequences; the sequences of the unlabeled bands were not obtained.
    Figure Legend Snippet: DGGE patterns of PCR-amplified 16S rDNA segments. Each lane represents a 100-μm vertical interval through the microbial mat. Bands 1 and 2 are cyanobacterial sequences; the bands labeled nc are noncyanobacterial sequences; the sequences of the unlabeled bands were not obtained.

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Labeling

    9) Product Images from "Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1"

    Article Title: Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042375

    Chromatin immunoprecipitation (ChIP) assays of SATB1 binding. A. Locations of primers (a–e) used for ChIP assays. B. ChIP analyses were carried out by using anti-mouse SATB1 antibody (left panel) or purified mouse IgG1 control antibody (central panel) jn HeLaS3 cells stably expressing SATB1. Chromain-immunoprecipitated DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. Error bars represent the mean and standard deviations based on four independent experiments. Relative ratio (SATB1/control) is shown as SATB1-specific binding (right panel).
    Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) assays of SATB1 binding. A. Locations of primers (a–e) used for ChIP assays. B. ChIP analyses were carried out by using anti-mouse SATB1 antibody (left panel) or purified mouse IgG1 control antibody (central panel) jn HeLaS3 cells stably expressing SATB1. Chromain-immunoprecipitated DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. Error bars represent the mean and standard deviations based on four independent experiments. Relative ratio (SATB1/control) is shown as SATB1-specific binding (right panel).

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Purification, Stable Transfection, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction

    10) Product Images from "IKK? and alternative NF-?B regulate PGC-1? to promote oxidative muscle metabolism"

    Article Title: IKK? and alternative NF-?B regulate PGC-1? to promote oxidative muscle metabolism

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201108118

    The alternative NF-κB subunit RelB regulates mitochondria and a glycolytic phenotype. (A) RNA was extracted from TA or soleus of 5-wk-old mice ( n = 4 each), and mitochondrial genes were quantitated by real-time PCR. Asterisks denote significance for COX IVi in TA (P = 0.04) and soleus (P = 0.0006) and COX Va in TA (P = 0.03) and soleus (P = 0.003). (B) Microarray analysis from soleus of adult RelB +/+ and RelB −/− littermates. Mitochondrial genes that are most down-regulated are shown. Green and red colors indicate lower and higher RNA expression, respectively. (C) Gene ontology classification of down-regulated genes in RelB −/− soleus muscles ( n = 3). Shown in parentheses is the number of genes that changes in each category. (D) Representative soleus from RelB +/+ and RelB −/− mice. (E) Total DNA was extracted from hind-limb muscles of 5-wk-old RelB +/+ and RelB −/− mice ( n = 3 each), and relative mitochondrial to nuclear copy numbers were calculated for TA (P = 0.04), soleus (P = 0.001), and gastrocnemius (Gas; P = 0.02). (F) TA muscles from 4-wk-old mice were harvested and stained for SDH. Bar, 200 µm. (G) RNA was prepared from soleus of RelB +/+ and RelB −/− adult mice as in A, and levels of myosin isotypes were determined by real-time RT-PCR. Asterisk denotes significance for MyHC IIa (P = 0.02) and MyHC IIb (P = 0.01). (H) TA homogenates from RelB +/+ and RelB −/− mice ( n = 4) were prepared and measured for lactate normalized to micrograms of muscle proteins. Asterisk denotes significance. (I) 4-wk-old RelB +/+ and RelB −/− mice were housed in individual metabolic cages for 72 h, and RER, total activity, and food intake were measured. Results shown are the mean of four mice per genotype, monitored during light and dark hours. *, P
    Figure Legend Snippet: The alternative NF-κB subunit RelB regulates mitochondria and a glycolytic phenotype. (A) RNA was extracted from TA or soleus of 5-wk-old mice ( n = 4 each), and mitochondrial genes were quantitated by real-time PCR. Asterisks denote significance for COX IVi in TA (P = 0.04) and soleus (P = 0.0006) and COX Va in TA (P = 0.03) and soleus (P = 0.003). (B) Microarray analysis from soleus of adult RelB +/+ and RelB −/− littermates. Mitochondrial genes that are most down-regulated are shown. Green and red colors indicate lower and higher RNA expression, respectively. (C) Gene ontology classification of down-regulated genes in RelB −/− soleus muscles ( n = 3). Shown in parentheses is the number of genes that changes in each category. (D) Representative soleus from RelB +/+ and RelB −/− mice. (E) Total DNA was extracted from hind-limb muscles of 5-wk-old RelB +/+ and RelB −/− mice ( n = 3 each), and relative mitochondrial to nuclear copy numbers were calculated for TA (P = 0.04), soleus (P = 0.001), and gastrocnemius (Gas; P = 0.02). (F) TA muscles from 4-wk-old mice were harvested and stained for SDH. Bar, 200 µm. (G) RNA was prepared from soleus of RelB +/+ and RelB −/− adult mice as in A, and levels of myosin isotypes were determined by real-time RT-PCR. Asterisk denotes significance for MyHC IIa (P = 0.02) and MyHC IIb (P = 0.01). (H) TA homogenates from RelB +/+ and RelB −/− mice ( n = 4) were prepared and measured for lactate normalized to micrograms of muscle proteins. Asterisk denotes significance. (I) 4-wk-old RelB +/+ and RelB −/− mice were housed in individual metabolic cages for 72 h, and RER, total activity, and food intake were measured. Results shown are the mean of four mice per genotype, monitored during light and dark hours. *, P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Microarray, RNA Expression, Staining, Quantitative RT-PCR, Activity Assay

    IKKα regulates mitochondrial biogenesis and fiber type specification in vivo. (A) Hind limbs from four independent litters of IKKα +/+ and IKKα −/− embryos (total n = 8) were isolated at E20. Cytochrome c and COX Va were measured by real-time quantitative RT-PCR. (B) Additional tissues from E20 limbs from A were homogenized and probed by Western blotting. (C) Total DNA was extracted from E20 limbs, and real-time RT-PCR analysis was performed to quantitate mitochondrial and nuclear (GAPDH) genome copy number. Results are means of five mice per genotype and calculated as fold mitochondrial over genomic DNA. P = 0.004. (D) AAV-GFP and AAV-IKKα viruses were injected into the TA. Mice were sacrificed after 3–4 mo, and protein lysates were prepared and analyzed by Western blotting. Shown is a representative blot from five independent experiments. (E and F) After similar viral injections as described in D, mitochondria were fractionated, and protein concentrations or DNA content was determined and normalized to total protein/DNA content (E; n = 5 per group; P
    Figure Legend Snippet: IKKα regulates mitochondrial biogenesis and fiber type specification in vivo. (A) Hind limbs from four independent litters of IKKα +/+ and IKKα −/− embryos (total n = 8) were isolated at E20. Cytochrome c and COX Va were measured by real-time quantitative RT-PCR. (B) Additional tissues from E20 limbs from A were homogenized and probed by Western blotting. (C) Total DNA was extracted from E20 limbs, and real-time RT-PCR analysis was performed to quantitate mitochondrial and nuclear (GAPDH) genome copy number. Results are means of five mice per genotype and calculated as fold mitochondrial over genomic DNA. P = 0.004. (D) AAV-GFP and AAV-IKKα viruses were injected into the TA. Mice were sacrificed after 3–4 mo, and protein lysates were prepared and analyzed by Western blotting. Shown is a representative blot from five independent experiments. (E and F) After similar viral injections as described in D, mitochondria were fractionated, and protein concentrations or DNA content was determined and normalized to total protein/DNA content (E; n = 5 per group; P

    Techniques Used: In Vivo, Isolation, Quantitative RT-PCR, Western Blot, Mouse Assay, Injection

    11) Product Images from "Clustered LAG-1 binding sites in lag-1/CSL are involved in regulating lag-1 expression during lin-12/Notch-dependent cell-fate specification"

    Article Title: Clustered LAG-1 binding sites in lag-1/CSL are involved in regulating lag-1 expression during lin-12/Notch-dependent cell-fate specification

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2013.46.4.269

    In vivo LAG-1 binding to a lag-1 regulatory region. ChIP was performed with the extracts from worms expressing SEL-8::GFP . PCR analysis was performed with primers described in MATERIALS AND METHODS. LAG-1 binding sites are in the first (1s st to 4 th LAG-1 binding sites in Fig. 1 , as well as in the in vitro DNA binding assay section in MATERIALS AND METHODS), third (5 th to 9 th ), and fourth (10 th to 13 th ) sections, but not in the second section. Pre-IP represents the input extracts subjected to IP. IgG and anti-HLH-2 antibodies were used as negative controls, and anti-LAG-3 and anti-GFP antibodies were used to detect the in vivo binding of LAG-1/LAG-3 complex to LAG-1 binding sites in lag-1 .
    Figure Legend Snippet: In vivo LAG-1 binding to a lag-1 regulatory region. ChIP was performed with the extracts from worms expressing SEL-8::GFP . PCR analysis was performed with primers described in MATERIALS AND METHODS. LAG-1 binding sites are in the first (1s st to 4 th LAG-1 binding sites in Fig. 1 , as well as in the in vitro DNA binding assay section in MATERIALS AND METHODS), third (5 th to 9 th ), and fourth (10 th to 13 th ) sections, but not in the second section. Pre-IP represents the input extracts subjected to IP. IgG and anti-HLH-2 antibodies were used as negative controls, and anti-LAG-3 and anti-GFP antibodies were used to detect the in vivo binding of LAG-1/LAG-3 complex to LAG-1 binding sites in lag-1 .

    Techniques Used: In Vivo, Binding Assay, Chromatin Immunoprecipitation, Expressing, Polymerase Chain Reaction, In Vitro, DNA Binding Assay

    12) Product Images from "Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus"

    Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007755

    Functional mapping of the LMOD1 locus identifies FOXO3 dependent mechanism at rs34091558. (A) Prediction of rs34091558 non-risk (TA) and risk allele (T) on FOXO3 binding based on JASPAR PWM scores. Human sequence (hg38) is shown aligned to the consensus FOXO3 sequence and mammalian genomic sequences and PhyloP conservation track. (B) Relative expression (RPKM) of candidate Forkhead transcription factors in coronary artery tissues (n = 133) from the GTEx dataset. (C) Luciferase activity examined for each of the LMOD1 enhancer constructs in A7r5 SMCs in the presence of FOXO3 . Results were reproduced in n = 3 independent experiments performed in quadruplicates. (D) Chromatin immunoprecipitation (ChIP) assay for LMOD1 and EGFR (as a positive control region) in HCASMC chromatin lysates immunoprecipitated with antibodies to FOXO3 or a negative control rabbit IgG. Results were repeated in n = 3 independent studies. (E) Allele specific ChIP (haploChIP) for FOXO3 protein in DNA derived from cultured HCASMC ChIP experiments. DNA from cell line homozygous for the ancestral allele was used as a positive control and arbitrarily set to 1. Values represent mean ± standard deviation of triplicates. Similar results were observed from n = 4 independent lines for each genotype. (F) Quantitative RT-PCR analysis of LMOD1 in HCASMCs following knock down of endogenous FOXO3 (F) or overexpression of FOXO3 (G). The results were reproduced in n = 3 independent experiments.
    Figure Legend Snippet: Functional mapping of the LMOD1 locus identifies FOXO3 dependent mechanism at rs34091558. (A) Prediction of rs34091558 non-risk (TA) and risk allele (T) on FOXO3 binding based on JASPAR PWM scores. Human sequence (hg38) is shown aligned to the consensus FOXO3 sequence and mammalian genomic sequences and PhyloP conservation track. (B) Relative expression (RPKM) of candidate Forkhead transcription factors in coronary artery tissues (n = 133) from the GTEx dataset. (C) Luciferase activity examined for each of the LMOD1 enhancer constructs in A7r5 SMCs in the presence of FOXO3 . Results were reproduced in n = 3 independent experiments performed in quadruplicates. (D) Chromatin immunoprecipitation (ChIP) assay for LMOD1 and EGFR (as a positive control region) in HCASMC chromatin lysates immunoprecipitated with antibodies to FOXO3 or a negative control rabbit IgG. Results were repeated in n = 3 independent studies. (E) Allele specific ChIP (haploChIP) for FOXO3 protein in DNA derived from cultured HCASMC ChIP experiments. DNA from cell line homozygous for the ancestral allele was used as a positive control and arbitrarily set to 1. Values represent mean ± standard deviation of triplicates. Similar results were observed from n = 4 independent lines for each genotype. (F) Quantitative RT-PCR analysis of LMOD1 in HCASMCs following knock down of endogenous FOXO3 (F) or overexpression of FOXO3 (G). The results were reproduced in n = 3 independent experiments.

    Techniques Used: Functional Assay, Binding Assay, Sequencing, Genomic Sequencing, Expressing, Luciferase, Activity Assay, Construct, Chromatin Immunoprecipitation, Positive Control, Immunoprecipitation, Negative Control, Derivative Assay, Cell Culture, Standard Deviation, Quantitative RT-PCR, Over Expression

    13) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

    14) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

    15) Product Images from "Collateral sensitivity constrains resistance evolution of the CTX-M-15 β-lactamase"

    Article Title: Collateral sensitivity constrains resistance evolution of the CTX-M-15 β-lactamase

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08529-y

    Frequency of CTX-M-15 clones selected on antibiotics and position of mutations. A blaCTX-M-15 gene mutant library obtained by error-prone PCR was grown on different antibiotic concentrations (0.5–4 × minimal inhibitory concentration (MIC)), the resulting clones were sequenced, and the percentage of reads for each SNP is shown for: mecillinam ( a ), piperacillin–tazobactam ( b ), and meropenem ( c ). The CTX-M-15 wild-type (CTX-M-15 WT) MIC for mecillinam is 0.25 µg/ml, for piperacillin–tazobactam is 16 µg/ml, and for meropenem is 0.06 µg/ml. d A 3D structure of the CTX-M-15-binding site with cefotaxime (orange) (PDB: 5FAP [ https://www.rcsb.org/structure/5FAP ]). Mutations providing high resistance against mecillinam: N135D (orange), against piperacillin–tazobactam: S133G and G239S (green), and amoxicillin–clavulanic acid (blue). Amino acid (AA) letters with uncharged side chains are written in black and negatively charged side chains in red
    Figure Legend Snippet: Frequency of CTX-M-15 clones selected on antibiotics and position of mutations. A blaCTX-M-15 gene mutant library obtained by error-prone PCR was grown on different antibiotic concentrations (0.5–4 × minimal inhibitory concentration (MIC)), the resulting clones were sequenced, and the percentage of reads for each SNP is shown for: mecillinam ( a ), piperacillin–tazobactam ( b ), and meropenem ( c ). The CTX-M-15 wild-type (CTX-M-15 WT) MIC for mecillinam is 0.25 µg/ml, for piperacillin–tazobactam is 16 µg/ml, and for meropenem is 0.06 µg/ml. d A 3D structure of the CTX-M-15-binding site with cefotaxime (orange) (PDB: 5FAP [ https://www.rcsb.org/structure/5FAP ]). Mutations providing high resistance against mecillinam: N135D (orange), against piperacillin–tazobactam: S133G and G239S (green), and amoxicillin–clavulanic acid (blue). Amino acid (AA) letters with uncharged side chains are written in black and negatively charged side chains in red

    Techniques Used: Clone Assay, Mutagenesis, Polymerase Chain Reaction, Concentration Assay, Binding Assay

    16) Product Images from "First report of molecular detection of fluoroquinolone resistance-associated gyrA mutations in multidrug-resistant clinical Mycobacterium tuberculosis isolates in Kuwait"

    Article Title: First report of molecular detection of fluoroquinolone resistance-associated gyrA mutations in multidrug-resistant clinical Mycobacterium tuberculosis isolates in Kuwait

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-123

    Species-specific identification of M. tuberculosis isolates by multiplex PCR . Representative agarose gel of amplicons of multiplex PCR from 7 selected multidrug-resistant M. tuberculosis isolates (lanes 1-7) showing M. tuberculosis -specific amplification of two fragments of 473 bp and 235 bp (marked by arrows) from oxyR and rpoB genes, respectively. Lane M is 100 bp DNA ladder and the position of migration of 100 and 600 bp fragments are marked.
    Figure Legend Snippet: Species-specific identification of M. tuberculosis isolates by multiplex PCR . Representative agarose gel of amplicons of multiplex PCR from 7 selected multidrug-resistant M. tuberculosis isolates (lanes 1-7) showing M. tuberculosis -specific amplification of two fragments of 473 bp and 235 bp (marked by arrows) from oxyR and rpoB genes, respectively. Lane M is 100 bp DNA ladder and the position of migration of 100 and 600 bp fragments are marked.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Migration

    17) Product Images from "High-Resolution Transcript Map of the Region Spanning D12S1629 and D12S312 at Chromosome 12q13: Triple A Syndrome-Linked Region"

    Article Title: High-Resolution Transcript Map of the Region Spanning D12S1629 and D12S312 at Chromosome 12q13: Triple A Syndrome-Linked Region

    Journal: Genome Research

    doi:

    An STS content map for the region spanning D12S1629 and D12S312. The line extending across the top represents the 12q13 region. The centromere is to the left and the telomere is to the right. Genetic distances in centimorgans (Généthon Genetic map) are indicated below the line. (A) A YAC map of the region. The map consists of 34 YAC clones and contains 58 STSs. Clones are from Center d'Etude du Polymorphisme Humain (CEPH) II YAC library. (B) A high-resolution transcript map. The map is composed of 217 BAC, PAC, and cosmid clones with 135 STSs. The order of most STSs was determined unequivocally, although that of a few STSs was not (depicted by brackets). The boxed clones represent minimum number of clones to represent the whole contig (minimal tiling path). Lines represent the clones and their names correspond to the plate addresses. Clones with B prefix; RPCI 11 BAC library, DB prefix; CIT-HSP BAC library, P prefix; RPCI 1 PAC library, C prefix; chromosome 12-specific cosmid library LL12NCO1. The STS content of each clone is indicated by symbols: black circle, genetic marker; blue square, random genomic marker; square with red border, STS derived from the clone-end sequence; red triangle pointing upward, gene; triangle pointing downward, ESTs; unfilled symbol, not confirmed by PCR.
    Figure Legend Snippet: An STS content map for the region spanning D12S1629 and D12S312. The line extending across the top represents the 12q13 region. The centromere is to the left and the telomere is to the right. Genetic distances in centimorgans (Généthon Genetic map) are indicated below the line. (A) A YAC map of the region. The map consists of 34 YAC clones and contains 58 STSs. Clones are from Center d'Etude du Polymorphisme Humain (CEPH) II YAC library. (B) A high-resolution transcript map. The map is composed of 217 BAC, PAC, and cosmid clones with 135 STSs. The order of most STSs was determined unequivocally, although that of a few STSs was not (depicted by brackets). The boxed clones represent minimum number of clones to represent the whole contig (minimal tiling path). Lines represent the clones and their names correspond to the plate addresses. Clones with B prefix; RPCI 11 BAC library, DB prefix; CIT-HSP BAC library, P prefix; RPCI 1 PAC library, C prefix; chromosome 12-specific cosmid library LL12NCO1. The STS content of each clone is indicated by symbols: black circle, genetic marker; blue square, random genomic marker; square with red border, STS derived from the clone-end sequence; red triangle pointing upward, gene; triangle pointing downward, ESTs; unfilled symbol, not confirmed by PCR.

    Techniques Used: Clone Assay, BAC Assay, Marker, Derivative Assay, Sequencing, Polymerase Chain Reaction

    18) Product Images from "Microchimeric fetal cells play a role in maternal wound healing after pregnancy"

    Article Title: Microchimeric fetal cells play a role in maternal wound healing after pregnancy

    Journal: Chimerism

    doi: 10.4161/chim.28746

    Figure 5. Nested PCR products of 345bp assessed by electrophoresis in a 1.2% Agarose gel. L = 100bp DNA ladder (Promega, UK), (2–6) CS-scars, (7) Positive control using placental tissue and (8) Negative control using nulliparous skin.
    Figure Legend Snippet: Figure 5. Nested PCR products of 345bp assessed by electrophoresis in a 1.2% Agarose gel. L = 100bp DNA ladder (Promega, UK), (2–6) CS-scars, (7) Positive control using placental tissue and (8) Negative control using nulliparous skin.

    Techniques Used: Nested PCR, Electrophoresis, Agarose Gel Electrophoresis, Positive Control, Negative Control

    19) Product Images from "Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase"

    Article Title: Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-4-15

    Map of sequence elements in the human rDNA repeat unit and identification of the BLM association domains. A. The human rDNA repeat unit (U13369) sequence features and the location of the region isolated with a polyclonal BLM antibody. The orange arrows indicate sites of primer sets. The dotted lines indicate the direction of DNA replication origins [ 30 ]. B. ChIP mapping of BLM association with the rDNA repeat and mapping of BLM domains required for rDNA association. Equivalent amounts of DNA recovered from the induced cell lines were amplified with Taq DNA polymerase and one of eight primer sets, denatured and transferred to a nylon membrane. The PCR products were hybridized with the radiolabeled forward primer in each set and quantitated using a phosphorimager. Hybridization units were calculated relative to the cell line expressing EGF alone. C. A simple model for the BLM-binding region of the rDNA repeat.
    Figure Legend Snippet: Map of sequence elements in the human rDNA repeat unit and identification of the BLM association domains. A. The human rDNA repeat unit (U13369) sequence features and the location of the region isolated with a polyclonal BLM antibody. The orange arrows indicate sites of primer sets. The dotted lines indicate the direction of DNA replication origins [ 30 ]. B. ChIP mapping of BLM association with the rDNA repeat and mapping of BLM domains required for rDNA association. Equivalent amounts of DNA recovered from the induced cell lines were amplified with Taq DNA polymerase and one of eight primer sets, denatured and transferred to a nylon membrane. The PCR products were hybridized with the radiolabeled forward primer in each set and quantitated using a phosphorimager. Hybridization units were calculated relative to the cell line expressing EGF alone. C. A simple model for the BLM-binding region of the rDNA repeat.

    Techniques Used: Sequencing, Isolation, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Hybridization, Expressing, Binding Assay

    20) Product Images from "Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells"

    Article Title: Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1152

    ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P
    Figure Legend Snippet: ( A ) Number of 8-oxodGs per million of dGs (8-oxodg/10 6 dG) measured by LC-MS/MS in untreated (NT), UV-irradiated (UV) and NAC-treated (NAC) MCF10A cells, as indicated. ( B ) Efficiency of polyclonal anti-8-oxodG from Millipore (Ab M), or monoclonal anti-8-oxodG from Trevigen (Ab T), and of anti-IgG antibodies in immuno-precipitation assays of 8-oxodG-containing synthetic ssDNA or G4 structures, as indicated. ( C ) Anti-8oxodG immuno-precipitation assay (% of input DNA, measured by qPCR; y axis) with equal amount (64 pg) of both synthetic oligonucleotides (8-oxodG-100mer and dG-100mer) added to 1 μg of NAC-treated genomic DNA. C1 and C2 indicate the same genomic negative control regions as in panel G. ( D ) Screenshot from the UCSC genome browser of 3.7 Mb from human chromosome 19 showing (top to bottom): OxiDIP-Seq signal profile, 8-oxodG peaks (dots), Input DNA, CG%, and RefSeq genes. ( E ) Screenshot from the UCSC genome browser of 2.1 Mb from human chromosome 19 showing OxiDIP-Seq signal profiles of two independent experiments (Exp #1 and #2) and Input DNA. ( F ) Scatter plot showing the correlation of the OxiDIP-Seq signals obtained in two independent experiments (Exp #1 and #2). Pearson's correlation coefficient (r), as indicated. Figure inset shows magnification of the high-density region. ( G ) OxiDIP-qPCR showing 8-oxodG enrichments (% of Input DNA) at eight different positive (#1–8) and two negative (C1, C2) regions, in untreated (black bar), UV- (gray), or NAC-treated (white) MCF10A cells. Screenshots from the UCSC genome browser show genomic position, 8-oxodG signal intensity, and qPCR probes (black box) of the selected regions. Data from two independent OxiDIP-qPCR assays are shown (±S.D.; P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Irradiation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    21) Product Images from "High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human"

    Article Title: High-resolution genome-wide functional dissection of transcriptional regulatory regions and nucleotides in human

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07746-1

    Overview of HiDRA. a Cells with the desired genotype and open chormatin patterns are selected for library construction. Tn5 transposase is used to preferentially fragment genomic DNA at regions of open chromatin. Fragments are then size-selected on an agarose gel and mtDNA contamination is removed by selective CRISPR-Cas9 degradation. The fragment library is amplified by PCR and cloned into an enhancer reporter vector. Gel image adapted from Buenrostro et al. 21 . Fragments are cloned into the STARR-seq vector backbone, introduced into target cells (which can differ from cells used to construct the library), and RNA is collected and sequenced. After data processing, the activity of partially-overlapping fragments is compared to identify driver nucleotides using the SHARPR-RE algorithm. b Size distribution of HiDRA library fragments (blue) and tiled regions (green). Bimodal shape for library fragment sizes is due to Tn5 preference to cut adjacent to nucleosomes. Fragment bin size = 20 nt, region bin size = 50 nt. c Number of ChromHMM-predicted active enhancer, active TSS and ATAC-seq peaks covered by multiple unique HiDRA fragments. d HiDRA plasmid library recapitulates the genomic coverage of a conventional ATAC-seq experiment
    Figure Legend Snippet: Overview of HiDRA. a Cells with the desired genotype and open chormatin patterns are selected for library construction. Tn5 transposase is used to preferentially fragment genomic DNA at regions of open chromatin. Fragments are then size-selected on an agarose gel and mtDNA contamination is removed by selective CRISPR-Cas9 degradation. The fragment library is amplified by PCR and cloned into an enhancer reporter vector. Gel image adapted from Buenrostro et al. 21 . Fragments are cloned into the STARR-seq vector backbone, introduced into target cells (which can differ from cells used to construct the library), and RNA is collected and sequenced. After data processing, the activity of partially-overlapping fragments is compared to identify driver nucleotides using the SHARPR-RE algorithm. b Size distribution of HiDRA library fragments (blue) and tiled regions (green). Bimodal shape for library fragment sizes is due to Tn5 preference to cut adjacent to nucleosomes. Fragment bin size = 20 nt, region bin size = 50 nt. c Number of ChromHMM-predicted active enhancer, active TSS and ATAC-seq peaks covered by multiple unique HiDRA fragments. d HiDRA plasmid library recapitulates the genomic coverage of a conventional ATAC-seq experiment

    Techniques Used: Agarose Gel Electrophoresis, CRISPR, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Construct, Activity Assay

    22) Product Images from "A Mouse Model of ?-Thalassemia Shows a Liver-Specific Down-Regulation of Abcc6 Expression"

    Article Title: A Mouse Model of ?-Thalassemia Shows a Liver-Specific Down-Regulation of Abcc6 Expression

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2010.10.004

    The transcription factors NFE2 and MTF-1 may be involved in the differential expression of Abcc6 in the liver of β-thalassemia mouse model ( Hbb th3/+ ) mice. Soluble chromatin prepared from the liver of wild-type (WT) ( A , upper panels ) and Hbb th3/+ mice ( A , lower panels ) was immunoprecipitated with an antibody raised against the indicated transcription factor. Negative control immunoprecipitations were performed with a nonimmunized control serum (NoAb). Chromatin preparations were analyzed for specific enrichment by PCR ( A ) and measured by quantitative PCR ( B ) using a pair of primers covering the promoter region of interest. For a positive control, chromatin extracts before immunoprecipitation (Input) was amplified in parallel. Standard errors are shown. * P
    Figure Legend Snippet: The transcription factors NFE2 and MTF-1 may be involved in the differential expression of Abcc6 in the liver of β-thalassemia mouse model ( Hbb th3/+ ) mice. Soluble chromatin prepared from the liver of wild-type (WT) ( A , upper panels ) and Hbb th3/+ mice ( A , lower panels ) was immunoprecipitated with an antibody raised against the indicated transcription factor. Negative control immunoprecipitations were performed with a nonimmunized control serum (NoAb). Chromatin preparations were analyzed for specific enrichment by PCR ( A ) and measured by quantitative PCR ( B ) using a pair of primers covering the promoter region of interest. For a positive control, chromatin extracts before immunoprecipitation (Input) was amplified in parallel. Standard errors are shown. * P

    Techniques Used: Expressing, Mouse Assay, Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Positive Control, Amplification

    23) Product Images from "In Vivo Expression of Salmonella enterica Serotype Typhi Genes in the Blood of Patients with Typhoid Fever in Bangladesh"

    Article Title: In Vivo Expression of Salmonella enterica Serotype Typhi Genes in the Blood of Patients with Typhoid Fever in Bangladesh

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001419

    Quantitative RT-PCR S. Typhi mRNA expression profiles in human blood compared to in vitro bacterial samples. Quantitative real time-PCR analysis of S. Typhi genes (A–H) comparing RNA recovered from blood of bacteremic patients to an in vitro culture of the corresponding clinical isolate. Genes represented in A–H were identified by SCOTS-cDNA as being more highly expressed in human blood samples than in i n vitro grown organisms; genes I–J had equivalent levels of detection in i n vivo versus in vitro samples by SCOTS. Mean copies of mRNA per copy of 16S rRNA and standard error of the mean are presented. * p
    Figure Legend Snippet: Quantitative RT-PCR S. Typhi mRNA expression profiles in human blood compared to in vitro bacterial samples. Quantitative real time-PCR analysis of S. Typhi genes (A–H) comparing RNA recovered from blood of bacteremic patients to an in vitro culture of the corresponding clinical isolate. Genes represented in A–H were identified by SCOTS-cDNA as being more highly expressed in human blood samples than in i n vitro grown organisms; genes I–J had equivalent levels of detection in i n vivo versus in vitro samples by SCOTS. Mean copies of mRNA per copy of 16S rRNA and standard error of the mean are presented. * p

    Techniques Used: Quantitative RT-PCR, Expressing, In Vitro, Real-time Polymerase Chain Reaction

    24) Product Images from "A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine"

    Article Title: A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042732

    Circulating HAdV types in southern Palestine. HAdV types circulating in Southern Palestine from 2005 to 2010 were detected using nested PCR and DNA sequencing assay of the HAdV hexon gene HVR1–6 followed by sequencing and sequence analysis. The 44 sequences were assigned the GenBank accession numbers [JQ796022–JQ796065]. The percentage of each serotype was calculated relatively to the number of total sequenced samples (44 samples).
    Figure Legend Snippet: Circulating HAdV types in southern Palestine. HAdV types circulating in Southern Palestine from 2005 to 2010 were detected using nested PCR and DNA sequencing assay of the HAdV hexon gene HVR1–6 followed by sequencing and sequence analysis. The 44 sequences were assigned the GenBank accession numbers [JQ796022–JQ796065]. The percentage of each serotype was calculated relatively to the number of total sequenced samples (44 samples).

    Techniques Used: Nested PCR, DNA Sequencing, Sequencing

    25) Product Images from "Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with the Linear Array HPV Genotyping PCR Assay and Influence of DNA Extraction Method on HPV Detection ▿Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with the Linear Array HPV Genotyping PCR Assay and Influence of DNA Extraction Method on HPV Detection ▿ ‡"

    Article Title: Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with the Linear Array HPV Genotyping PCR Assay and Influence of DNA Extraction Method on HPV Detection ▿Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with the Linear Array HPV Genotyping PCR Assay and Influence of DNA Extraction Method on HPV Detection ▿ ‡

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00235-10

    Multiplex HPV PCR versus the standard Linear Array with Qiagen MinElute media kit DNA extraction.
    Figure Legend Snippet: Multiplex HPV PCR versus the standard Linear Array with Qiagen MinElute media kit DNA extraction.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, DNA Extraction

    26) Product Images from "Dysregulated immune system networks in war veterans with PTSD is an outcome of altered miRNA expression and DNA methylation"

    Article Title: Dysregulated immune system networks in war veterans with PTSD is an outcome of altered miRNA expression and DNA methylation

    Journal: Scientific Reports

    doi: 10.1038/srep31209

    DNA methylation level has a trend that corroborates gene expression. There is a clear trend showing higher DNA methylation and lowered mRNA levels and vice-versa for the corresponding gene. (a) DNA methylation levels of the select genes presented as box plot. On x-axis, the names of gene are provided and y-axis provides the average β- values of DNA methylation. The two bars corresponding to each gene represent the DNA methylation level for control followed by PTSD patient in a left to right direction. (b) Transcript levels (y-axis: log 2 fold change values) of genes, after RNA-Seq analysis, listed in Fig. 4a. (c) Real time PCR validation of differentially expressed genes. To validate the RNA-Seq results, qRT-PCR was performed for seven representative genes with cDNA prepared from total RNA obtained from PBMCs of 24 control and 24 PTSD patients. The values are relative abundance (RA) values after qRT-PCR. The table inside the figure provides log 2 fold change values of the respective genes after RNA-Seq analysis. The error bars indicate standard error.
    Figure Legend Snippet: DNA methylation level has a trend that corroborates gene expression. There is a clear trend showing higher DNA methylation and lowered mRNA levels and vice-versa for the corresponding gene. (a) DNA methylation levels of the select genes presented as box plot. On x-axis, the names of gene are provided and y-axis provides the average β- values of DNA methylation. The two bars corresponding to each gene represent the DNA methylation level for control followed by PTSD patient in a left to right direction. (b) Transcript levels (y-axis: log 2 fold change values) of genes, after RNA-Seq analysis, listed in Fig. 4a. (c) Real time PCR validation of differentially expressed genes. To validate the RNA-Seq results, qRT-PCR was performed for seven representative genes with cDNA prepared from total RNA obtained from PBMCs of 24 control and 24 PTSD patients. The values are relative abundance (RA) values after qRT-PCR. The table inside the figure provides log 2 fold change values of the respective genes after RNA-Seq analysis. The error bars indicate standard error.

    Techniques Used: DNA Methylation Assay, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    27) Product Images from "EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma"

    Article Title: EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa901

    EWS-FLI1 and CRC TFs co-operatively activate the transcription of KLF15. ( A ) Integrative Genomics Viewer (IGV) plots of ChIP-Seq showing co-occupancy of EWS-FLI1 and CRC TFs at the super-enhancer and promoter of the KLF15 gene locus. The tracks were showing the overlapping signals of three primary Ewing sarcoma tissues and four Ewing sarcoma cell lines. Hi-C interactions were re-analyzed from the Hi-C data of SKNMC cell line downloaded from ENCODE database; H3K27ac, H3K4me3 and EWS-FLI1 ChIP-Seq data were retrieved from GEO (GSE61953). ( B ) ATAC-Seq and ChIP-Seq profiles at KLF15 super-enhancer region in either the presence or absence of EWS-FLI1 overexpression (top 4 tracks) or knockdown (bottom 4 tracks). Data were retrieved from GEO (GSE61953). ( C ) Zoom in view of ChIP-Seq signals in KLF15 locus. Three candidate enhancer elements (CE1-CE3), two promoter reigns (PR1, PR2) and one negative control region were separately cloned into luciferase reporter vectors. ( D ) Enhancer and ( E ) promoter activities were measured by luciferase reporter assays in A673 cells in either the presence or absence of knockdown of indicated TFs. ( F ) ChIP-qPCR using H3K27ac antibody was performed on A673 cells expressing either dCas9/KRAB vector alone or together with sgRNAs targeting CE1 and CE2. IgG was used as a negative control antibody. ( G ) qRT-PCR measuring the mRNA expression of CRC TFs in indicated groups. Mean ± s.d. are shown, Two-way ANOVA Dunnett's multiple comparisons test. n = 3. * P
    Figure Legend Snippet: EWS-FLI1 and CRC TFs co-operatively activate the transcription of KLF15. ( A ) Integrative Genomics Viewer (IGV) plots of ChIP-Seq showing co-occupancy of EWS-FLI1 and CRC TFs at the super-enhancer and promoter of the KLF15 gene locus. The tracks were showing the overlapping signals of three primary Ewing sarcoma tissues and four Ewing sarcoma cell lines. Hi-C interactions were re-analyzed from the Hi-C data of SKNMC cell line downloaded from ENCODE database; H3K27ac, H3K4me3 and EWS-FLI1 ChIP-Seq data were retrieved from GEO (GSE61953). ( B ) ATAC-Seq and ChIP-Seq profiles at KLF15 super-enhancer region in either the presence or absence of EWS-FLI1 overexpression (top 4 tracks) or knockdown (bottom 4 tracks). Data were retrieved from GEO (GSE61953). ( C ) Zoom in view of ChIP-Seq signals in KLF15 locus. Three candidate enhancer elements (CE1-CE3), two promoter reigns (PR1, PR2) and one negative control region were separately cloned into luciferase reporter vectors. ( D ) Enhancer and ( E ) promoter activities were measured by luciferase reporter assays in A673 cells in either the presence or absence of knockdown of indicated TFs. ( F ) ChIP-qPCR using H3K27ac antibody was performed on A673 cells expressing either dCas9/KRAB vector alone or together with sgRNAs targeting CE1 and CE2. IgG was used as a negative control antibody. ( G ) qRT-PCR measuring the mRNA expression of CRC TFs in indicated groups. Mean ± s.d. are shown, Two-way ANOVA Dunnett's multiple comparisons test. n = 3. * P

    Techniques Used: Chromatin Immunoprecipitation, Hi-C, Over Expression, Negative Control, Clone Assay, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Quantitative RT-PCR

    28) Product Images from "Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing"

    Article Title: Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17029-3

    5A2-DOT-X LNPs simplify generation of complex mouse models. a To create an in situ liver-specific cancer model, 5A2-DOT-5 LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs were injected into adult C57BL/6 mice weekly (three injections, 2.5 mg kg −1 total sgRNA, IV, n = 4). After 12, 15, and 20 weeks, mice were sacrificed and livers were collected to analyze tumor generation. b T7EI cleavage results from genomic DNA extracted from livers confirmed gene editing occurred at all three loci. Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. c Representative photograph of a mouse liver containing tumors excised 20 weeks after injection. d H E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in tumor lesions. Scale bar = 100 μm. e To create an in situ lung-specific cancer model, 5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlk RNPs were injected into adult C57BL/6 mice once (2 mg kg −1 ) or twice (1.5 mg kg −1 weekly for 2 weeks) (IV, n = 5). After 10, 16, and 24 weeks, mice were sacrificed and lungs were collected to analyze tumor generation. f T7EI cleavage results from genomic DNA extracted from lungs confirmed gene editing occurred at loci of Eml4 and Alk . Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. g PCR amplicons of Eml4-Alk rearrangements were also detected in all lungs treated with 5A2-DOT-50 LNPs. h Eml4-Alk rearrangements were further confirmed by sub-cloning and DNA sequencing. i H E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in lung tumor lesions. Scale bar = 100 μm. Data of b , d , f , g , and i were repeated three times independently with similar results.
    Figure Legend Snippet: 5A2-DOT-X LNPs simplify generation of complex mouse models. a To create an in situ liver-specific cancer model, 5A2-DOT-5 LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs were injected into adult C57BL/6 mice weekly (three injections, 2.5 mg kg −1 total sgRNA, IV, n = 4). After 12, 15, and 20 weeks, mice were sacrificed and livers were collected to analyze tumor generation. b T7EI cleavage results from genomic DNA extracted from livers confirmed gene editing occurred at all three loci. Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. c Representative photograph of a mouse liver containing tumors excised 20 weeks after injection. d H E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in tumor lesions. Scale bar = 100 μm. e To create an in situ lung-specific cancer model, 5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlk RNPs were injected into adult C57BL/6 mice once (2 mg kg −1 ) or twice (1.5 mg kg −1 weekly for 2 weeks) (IV, n = 5). After 10, 16, and 24 weeks, mice were sacrificed and lungs were collected to analyze tumor generation. f T7EI cleavage results from genomic DNA extracted from lungs confirmed gene editing occurred at loci of Eml4 and Alk . Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. g PCR amplicons of Eml4-Alk rearrangements were also detected in all lungs treated with 5A2-DOT-50 LNPs. h Eml4-Alk rearrangements were further confirmed by sub-cloning and DNA sequencing. i H E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in lung tumor lesions. Scale bar = 100 μm. Data of b , d , f , g , and i were repeated three times independently with similar results.

    Techniques Used: In Situ, Injection, Mouse Assay, Sequencing, Staining, Biomarker Assay, Expressing, Polymerase Chain Reaction, Subcloning, DNA Sequencing

    29) Product Images from "Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged Single-Stranded Adeno-Associated Viral Genomes in Academic Settings"

    Article Title: Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged Single-Stranded Adeno-Associated Viral Genomes in Academic Settings

    Journal: Human Gene Therapy Methods

    doi: 10.1089/hgtb.2019.110

    Overview of Fast-Seq, a Tn5-based packaged ssAAV genome sequencing method. Preparations of rAAV undergo ssDNA extraction, second-strand synthesis to generate dsDNA, tagmentation with adapter-loaded Tn5 transposomes, indexing, QC validation, short-read sequencing on Illumina MiSeq, mapping reads to the transfer vector plasmid used to generate the input rAAV, and sequence validation of the packaged genome. An example packaged genome mutation (*) is shown at position 7. dsAAV, double-stranded AAV; dsDNA, double-stranded DNA; rAAV, recombinant AAV; ssAAV, single-stranded adeno-associated virus; Tn5, transposase.
    Figure Legend Snippet: Overview of Fast-Seq, a Tn5-based packaged ssAAV genome sequencing method. Preparations of rAAV undergo ssDNA extraction, second-strand synthesis to generate dsDNA, tagmentation with adapter-loaded Tn5 transposomes, indexing, QC validation, short-read sequencing on Illumina MiSeq, mapping reads to the transfer vector plasmid used to generate the input rAAV, and sequence validation of the packaged genome. An example packaged genome mutation (*) is shown at position 7. dsAAV, double-stranded AAV; dsDNA, double-stranded DNA; rAAV, recombinant AAV; ssAAV, single-stranded adeno-associated virus; Tn5, transposase.

    Techniques Used: Sequencing, Plasmid Preparation, Mutagenesis, Recombinant

    30) Product Images from "Distinct Roles of Gαq and Gα11 for Purkinje Cell Signaling and Motor Behavior"

    Article Title: Distinct Roles of Gαq and Gα11 for Purkinje Cell Signaling and Motor Behavior

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4193-03.2004

    Real-time quantitative RT-PCR of Gα q and Gα 11 transcripts in mutant and wild-type mice. A , Quantification of Gα q and Gα 11 transcripts in whole brain from wild-type, Gα q -deficient, and Gα 11 -deficient mice. Left, Agarose gel electrophoresis of representative examples of cDNAs obtained after PCR amplification (Gα q amplicons, 724-for/935-rev, 212 bp; Gα 11 amplicons, 119-for/403-rev, 285 bp). Standards: Gα q , 2000 cDNA copies; Gα 11 , 50 cDNA copies were amplified; the marker is the MassRuler DNA Ladder (Fermentas, St. Leon-Rot, Germany). Right, Bar graph illustrating the average number of cDNA copies per nanogram of total brain RNA. n = 6 mRNA pools from two animals; n.d., Not detectable. B , Quantification of the Gα q and Gα 11 isoforms in single Purkinje cells from wild-type, Gα q -deficient, and Gα 11 -deficient mice. Left, Agarose gel electrophoresis of the cDNA obtained after PCR amplification (above: Gα q amplicons, 86-for/256-rev, 171 bp; below: Gα 11 amplicons, 119-for/403-rev, 285 bp). Standards: Gα q , 50 copies; Gα 11 , 8 copies were amplified; marker (see A ); ACSF was harvested directly above the slice; RT–is a Purkinje cell control without reverse transcriptase enzyme added; cells 1–3 are from wild-type animals. Cell 3 shows an example of a single cell that was positive for Gα q but negative for Gα 11 . Right, Bar graph illustrating the average cDNA copy number per single Purkinje cell; copy numbers are determined based on the standard curves (see C ). C , Real-time fluorescence measurements of the amplification of the Gα q (top) and Gα 11 cDNA (bottom). Left, For the absolute quantification of cDNAs, known dilutions of the corresponding amplicons were amplified and used to form standard curves. Right, Representative examples of the PCR amplification of single Purkinje cells from wild-type mice ( n = 10 cells); only one of five for Gα q or two of five for Gα 11 of a Purkinje cell was used for amplification.
    Figure Legend Snippet: Real-time quantitative RT-PCR of Gα q and Gα 11 transcripts in mutant and wild-type mice. A , Quantification of Gα q and Gα 11 transcripts in whole brain from wild-type, Gα q -deficient, and Gα 11 -deficient mice. Left, Agarose gel electrophoresis of representative examples of cDNAs obtained after PCR amplification (Gα q amplicons, 724-for/935-rev, 212 bp; Gα 11 amplicons, 119-for/403-rev, 285 bp). Standards: Gα q , 2000 cDNA copies; Gα 11 , 50 cDNA copies were amplified; the marker is the MassRuler DNA Ladder (Fermentas, St. Leon-Rot, Germany). Right, Bar graph illustrating the average number of cDNA copies per nanogram of total brain RNA. n = 6 mRNA pools from two animals; n.d., Not detectable. B , Quantification of the Gα q and Gα 11 isoforms in single Purkinje cells from wild-type, Gα q -deficient, and Gα 11 -deficient mice. Left, Agarose gel electrophoresis of the cDNA obtained after PCR amplification (above: Gα q amplicons, 86-for/256-rev, 171 bp; below: Gα 11 amplicons, 119-for/403-rev, 285 bp). Standards: Gα q , 50 copies; Gα 11 , 8 copies were amplified; marker (see A ); ACSF was harvested directly above the slice; RT–is a Purkinje cell control without reverse transcriptase enzyme added; cells 1–3 are from wild-type animals. Cell 3 shows an example of a single cell that was positive for Gα q but negative for Gα 11 . Right, Bar graph illustrating the average cDNA copy number per single Purkinje cell; copy numbers are determined based on the standard curves (see C ). C , Real-time fluorescence measurements of the amplification of the Gα q (top) and Gα 11 cDNA (bottom). Left, For the absolute quantification of cDNAs, known dilutions of the corresponding amplicons were amplified and used to form standard curves. Right, Representative examples of the PCR amplification of single Purkinje cells from wild-type mice ( n = 10 cells); only one of five for Gα q or two of five for Gα 11 of a Purkinje cell was used for amplification.

    Techniques Used: Quantitative RT-PCR, Mutagenesis, Mouse Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Fluorescence

    31) Product Images from "A retrospective study of community-acquired Clostridium difficile infection in southwest China"

    Article Title: A retrospective study of community-acquired Clostridium difficile infection in southwest China

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21762-7

    The toxin profiles and PCR ribotyping characters of C. difficile between children and adults in this study. ( A ) The toxin profile of isolated strains. ( B ) RT constituent ratio of C. difficile strains. Left: children group; right: adults group. ( C ) RT distribu tion characters of C. difficile for different hospitals in children group. ( D ) RT distribution characters of C. difficile for different hospitals in adults group. ( E ). RT distribution characters of C. difficile for fecal property in children group. ( F ) RT distribution characters of C. difficile for fecal property in adults group.
    Figure Legend Snippet: The toxin profiles and PCR ribotyping characters of C. difficile between children and adults in this study. ( A ) The toxin profile of isolated strains. ( B ) RT constituent ratio of C. difficile strains. Left: children group; right: adults group. ( C ) RT distribu tion characters of C. difficile for different hospitals in children group. ( D ) RT distribution characters of C. difficile for different hospitals in adults group. ( E ). RT distribution characters of C. difficile for fecal property in children group. ( F ) RT distribution characters of C. difficile for fecal property in adults group.

    Techniques Used: Polymerase Chain Reaction, Isolation

    32) Product Images from "SABER enables amplified and multiplexed imaging of RNA and DNA in cells and tissues"

    Article Title: SABER enables amplified and multiplexed imaging of RNA and DNA in cells and tissues

    Journal: Nature methods

    doi: 10.1038/s41592-019-0404-0

    Sequential imaging of chromosomal targets using Exchange-SABER. a , Schematic of 17 targeted regions along the human X chromosome (width to scale). Each set of probes per spot had different 42mer barcode sequences appended to their 3’ ends ( Fig. 1d ). Seventeen 42mer bridge sequences concatemerized with 17 different PER primers were co-hybridized. b , Individual color channels on DAPI. 6 hybridizations, targeting 3, 3, 3, 3, 3, and 2 spots, respectively, that took course over a single day were used to image the 17 colors. c , 17-color overlays on DAPI. The representative metaphase spread from part (B) is shown overlaid on DAPI at two length scales (top left, top right). Interphase cells showing the X chromosome territories were also captured (bottom). d , Combinatorial 6-color SABER imaging. As a step toward increasing multiplexing with SABER amplification further, we demonstrated mapping six of the spots on the chromosome to 4 different 6-color combinations. Scale bars: 5 μm (spreads), 20 μm (fields of view).
    Figure Legend Snippet: Sequential imaging of chromosomal targets using Exchange-SABER. a , Schematic of 17 targeted regions along the human X chromosome (width to scale). Each set of probes per spot had different 42mer barcode sequences appended to their 3’ ends ( Fig. 1d ). Seventeen 42mer bridge sequences concatemerized with 17 different PER primers were co-hybridized. b , Individual color channels on DAPI. 6 hybridizations, targeting 3, 3, 3, 3, 3, and 2 spots, respectively, that took course over a single day were used to image the 17 colors. c , 17-color overlays on DAPI. The representative metaphase spread from part (B) is shown overlaid on DAPI at two length scales (top left, top right). Interphase cells showing the X chromosome territories were also captured (bottom). d , Combinatorial 6-color SABER imaging. As a step toward increasing multiplexing with SABER amplification further, we demonstrated mapping six of the spots on the chromosome to 4 different 6-color combinations. Scale bars: 5 μm (spreads), 20 μm (fields of view).

    Techniques Used: Imaging, Multiplexing, Amplification

    33) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

    34) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

    35) Product Images from "Regulation of Mammalian Epithelial Differentiation and Intestine Development by Class I Histone Deacetylases"

    Article Title: Regulation of Mammalian Epithelial Differentiation and Intestine Development by Class I Histone Deacetylases

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.8.3132-3139.2004

    Chemical HDAC inhibitors induce epithelial differentiation in mouse fetal gut explants. (A) Immunoblots showing hyperacetylation of histones H3 and H4 in E13 explants treated with 3 mM VPA or 2 mM SB for 24 h and reduced HDAC2 levels in response to VPA. (B) Effects of VPA and SB on expression of molecular markers of epithelial differentiation, as analyzed by RT-PCR, in E13 gut explants cultured for 48 or 72 h after chemical exposure. Apo1a, apolipoprotein 1A; Fabpi and Fabpl, intestinal and liver fatty acid-binding proteins 1 and 2, respectively; Upa, uterine-specific proline-rich acidic protein; Mt2, metallothionein 2; HDGF, hepatoma-derived growth factor;Gapdh, glyceraldehyde 3-phosphate dehydrogenase (loading control). Each lane includes two pooled explants, and the results are representative of five independent experiments. (C) Accelerated appearance of villiform epithelium in E13 gut explants treated with VPA and cultured for 4 days. (D) Dose-dependent induction of the representative differentiation marker Apo1a by VPA and SB. (E) ChIP analysis shows increased association of acetylated histones H3 and H4 with promoters of the Apo1a , Fabpl , Fabpi , and Mt2 genes following injection of 3 mM VPA into the E13 gut lumen and 24-h culture. PCR was performed with a radiotracer, and input DNA for PCR (Pre-IP) was 1/50 the amount used for immunoprecipitation; chromatin subjected to the same treatment without antibody served as the negative control. Each lane contains samples pooled from six to seven gut explants, and the results are representative of three separate experiments with identical results.
    Figure Legend Snippet: Chemical HDAC inhibitors induce epithelial differentiation in mouse fetal gut explants. (A) Immunoblots showing hyperacetylation of histones H3 and H4 in E13 explants treated with 3 mM VPA or 2 mM SB for 24 h and reduced HDAC2 levels in response to VPA. (B) Effects of VPA and SB on expression of molecular markers of epithelial differentiation, as analyzed by RT-PCR, in E13 gut explants cultured for 48 or 72 h after chemical exposure. Apo1a, apolipoprotein 1A; Fabpi and Fabpl, intestinal and liver fatty acid-binding proteins 1 and 2, respectively; Upa, uterine-specific proline-rich acidic protein; Mt2, metallothionein 2; HDGF, hepatoma-derived growth factor;Gapdh, glyceraldehyde 3-phosphate dehydrogenase (loading control). Each lane includes two pooled explants, and the results are representative of five independent experiments. (C) Accelerated appearance of villiform epithelium in E13 gut explants treated with VPA and cultured for 4 days. (D) Dose-dependent induction of the representative differentiation marker Apo1a by VPA and SB. (E) ChIP analysis shows increased association of acetylated histones H3 and H4 with promoters of the Apo1a , Fabpl , Fabpi , and Mt2 genes following injection of 3 mM VPA into the E13 gut lumen and 24-h culture. PCR was performed with a radiotracer, and input DNA for PCR (Pre-IP) was 1/50 the amount used for immunoprecipitation; chromatin subjected to the same treatment without antibody served as the negative control. Each lane contains samples pooled from six to seven gut explants, and the results are representative of three separate experiments with identical results.

    Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Binding Assay, Derivative Assay, Marker, Chromatin Immunoprecipitation, Injection, Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    36) Product Images from "Conservative site-specific and single-copy transgenesis in human LINE-1 elements"

    Article Title: Conservative site-specific and single-copy transgenesis in human LINE-1 elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1345

    Targeting endogenous att H4X in HT1080 cells. ( A ) Diagram showing the 21 nucleotide sequences comprising the core binding and spacer sequences of various att sites and features of the LINE -1 retrotransposon. Wild type att B and att P sequences aligned to show the respective core binding and spacer sequences. The query sequence was used in a bioinformatics search for targets resembling core att sites. Sequence logo analysis was performed for the 18 bp att H4X sequences in LINE-1 elements in the targeted cell lines including additional three nucleotides at the 5′ end in order to compare with the 21 bp att B sequence. A LINE -1 retrotransposon diagram with the position and orientation of att H4X is shown at the bottom. See text for details. ( B ) Schematic drawing showing pattP4X-PGKssPuro target vector and predicted recombination between att P4X and genomic att H4X. Positions of relevant primers (Puro rev24, Puro rev303, Puro fw318, Puro fw509 and pr21), restriction sites and the probe used for Southern blotting are indicated. ( C ) Screening for att H4X × att P4X recombination events in HT1080 clones. PCR was performed with genomic DNA and primers cs_ att H4X_F1 and Puro rev24 (for att L junction) and cs_ att H4X_R1 and pr21 (for att R junction). PCR amplified products of the expected size (1100 bp; for the left junction) were detected in clones 3, 19 and 21 (top two panels) and (∼375 bp; for the right junction) in clones 11, 19 and 21 (bottom two panels). W, no DNA template control; HT, negative control (genomic DNA from parental cells); Ina, genomic DNA from puromycin resistant clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssIna , the latter expressed inactive Int as negative control; +, positive control (genomic DNA from a HT1080 clone carrying an att H4X x att P4X integration event); M, 100 bp DNA ladder; 1 to 33, genomic DNA from puromycin resistant HT1080 clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from five targeted HT1080 clones, as indicated, and parental HT1080 cell line was subjected to digestion with PstI or XbaI. A PCR-derived digoxigenin-labeled probe complementary to the puromycin resistance gene was used. Lanes: M1, 1 kb DNA ladder; HT, genomic DNA from HT1080; 3, 11, 18*, 19 and 21, genomic DNA from targeted clones; pP4X (10 7 , 10 8 , 10 9 ), copies of linearized pattP4X-PGKssPuro loaded as positive control. The arrows indicate fragments of expected size for clones 3 and 11. *HT 1080 Clone 18 (carrying a single-copy transgene) was obtained from a screen of puromycin-resistant HT1080 clones that resulted from a previous co-transfection of pattP4X-PGKssPuro and pCMVssInt-h/218 . White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones.
    Figure Legend Snippet: Targeting endogenous att H4X in HT1080 cells. ( A ) Diagram showing the 21 nucleotide sequences comprising the core binding and spacer sequences of various att sites and features of the LINE -1 retrotransposon. Wild type att B and att P sequences aligned to show the respective core binding and spacer sequences. The query sequence was used in a bioinformatics search for targets resembling core att sites. Sequence logo analysis was performed for the 18 bp att H4X sequences in LINE-1 elements in the targeted cell lines including additional three nucleotides at the 5′ end in order to compare with the 21 bp att B sequence. A LINE -1 retrotransposon diagram with the position and orientation of att H4X is shown at the bottom. See text for details. ( B ) Schematic drawing showing pattP4X-PGKssPuro target vector and predicted recombination between att P4X and genomic att H4X. Positions of relevant primers (Puro rev24, Puro rev303, Puro fw318, Puro fw509 and pr21), restriction sites and the probe used for Southern blotting are indicated. ( C ) Screening for att H4X × att P4X recombination events in HT1080 clones. PCR was performed with genomic DNA and primers cs_ att H4X_F1 and Puro rev24 (for att L junction) and cs_ att H4X_R1 and pr21 (for att R junction). PCR amplified products of the expected size (1100 bp; for the left junction) were detected in clones 3, 19 and 21 (top two panels) and (∼375 bp; for the right junction) in clones 11, 19 and 21 (bottom two panels). W, no DNA template control; HT, negative control (genomic DNA from parental cells); Ina, genomic DNA from puromycin resistant clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssIna , the latter expressed inactive Int as negative control; +, positive control (genomic DNA from a HT1080 clone carrying an att H4X x att P4X integration event); M, 100 bp DNA ladder; 1 to 33, genomic DNA from puromycin resistant HT1080 clones obtained through co-transfection of pattP4X-PGKssPuro and pCMVssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from five targeted HT1080 clones, as indicated, and parental HT1080 cell line was subjected to digestion with PstI or XbaI. A PCR-derived digoxigenin-labeled probe complementary to the puromycin resistance gene was used. Lanes: M1, 1 kb DNA ladder; HT, genomic DNA from HT1080; 3, 11, 18*, 19 and 21, genomic DNA from targeted clones; pP4X (10 7 , 10 8 , 10 9 ), copies of linearized pattP4X-PGKssPuro loaded as positive control. The arrows indicate fragments of expected size for clones 3 and 11. *HT 1080 Clone 18 (carrying a single-copy transgene) was obtained from a screen of puromycin-resistant HT1080 clones that resulted from a previous co-transfection of pattP4X-PGKssPuro and pCMVssInt-h/218 . White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones.

    Techniques Used: Binding Assay, Sequencing, Plasmid Preparation, Southern Blot, Clone Assay, Polymerase Chain Reaction, Amplification, Negative Control, Cotransfection, Positive Control, Purification, Derivative Assay, Labeling

    Targeting att H4X in hESCs with pattP4X-PGKssPuro-UTF1-eGFP . ( A ) Schematic diagram of pattP4X-PGKssPuro-UTF1-eGFP targeting vector after integration. Locations of primers (Puro rev24, PGK rev, pr111 and pr22) and the Southern probe are indicated. ( B ) Screening for att H4X x att P4X recombination events in selected hESC clones. Semi-nested PCR with primers cs_ att H4X_F1 and PGK rev (for the left junction) using templates obtained with primary PCR (primers cs_ att H4X_F1 and Puro rev24). PCR products of the expected size (∼900 bp) were detected in hESC clone E3 (left panel). Confirmatory PCR with genomic locus specific primers were performed for clone E3. PCR products of expected size (∼1100 bp) were obtained in a semi-nested PCR with primer PGK rev and genomic locus-specific forward primer E3-GF2 using templates from a primary PCR (primers Puro rev24 and primer E3-GF2; middle panel). PCR products of expected size (∼1000 bp) were obtained in a semi-nested PCR with primer pr111 and genomic locus specific reverse primer E3-GR2 (for the right junction) using templates from a primary PCR (primers pr22 and E3-GR2; right panel). W, no DNA template control; ES, negative control (genomic DNA from parental cells); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; E2, E3, G1, G9, H2, J4, B16, A20, genomic DNA from puromycin resistant and GFP-positive hESC clones obtained through co-transfection of pattP4X-PGKssPuro-UTF1-eGFP and pEF1α-ss-Int-C3CNLS . ( C ) Southern blot analysis. Genomic DNA from three hESC clones and parental hESC cells were digested with NdeI or XbaI. Digoxigenin-labeled probe to EGFP was employed. Lanes: M1, 1 kb DNA ladder; 10 8 , 10 9 , copies of linearized targeting vector as positive control; ES, parental DNA; A3, E3 and K3, genomic DNA from targeted hESC clones. White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones. ( D ) Functional test for UTF1 promoter-driven EGFP expression in targeted hESC clones. Fluorescence microscopic analysis of undifferentiated and RA-induced, differentiated parental hES-047 cells and clones A3, E3 and K3. EGFP expression was detected with the undifferentiated hESC clones A3, E3 and K3 (column 2, panels 2, 3 and 4) but disappeared in differentiated progenies (column 4, panels 2, 3 and 4) respectively. Panels in columns 1 and 3 are phase-contrast light micrographs of undifferentiated and differentiated cells, respectively. Magnification 5×; Scale bars 100 μm. ( E ) FACS analysis for undifferentiated and differentiated hESCs clones Dot plots representing GFP + cells (upper right quadrant) and GFP − cells (lower right quadrant) for the untargeted hESCs, undifferentiated targeted hESC clones (A3, E3, K3) after 3 weeks (early) and 8 weeks (late) of culturing the cells (left and middle panel) and their differentiated progenies (right panel).
    Figure Legend Snippet: Targeting att H4X in hESCs with pattP4X-PGKssPuro-UTF1-eGFP . ( A ) Schematic diagram of pattP4X-PGKssPuro-UTF1-eGFP targeting vector after integration. Locations of primers (Puro rev24, PGK rev, pr111 and pr22) and the Southern probe are indicated. ( B ) Screening for att H4X x att P4X recombination events in selected hESC clones. Semi-nested PCR with primers cs_ att H4X_F1 and PGK rev (for the left junction) using templates obtained with primary PCR (primers cs_ att H4X_F1 and Puro rev24). PCR products of the expected size (∼900 bp) were detected in hESC clone E3 (left panel). Confirmatory PCR with genomic locus specific primers were performed for clone E3. PCR products of expected size (∼1100 bp) were obtained in a semi-nested PCR with primer PGK rev and genomic locus-specific forward primer E3-GF2 using templates from a primary PCR (primers Puro rev24 and primer E3-GF2; middle panel). PCR products of expected size (∼1000 bp) were obtained in a semi-nested PCR with primer pr111 and genomic locus specific reverse primer E3-GR2 (for the right junction) using templates from a primary PCR (primers pr22 and E3-GR2; right panel). W, no DNA template control; ES, negative control (genomic DNA from parental cells); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; E2, E3, G1, G9, H2, J4, B16, A20, genomic DNA from puromycin resistant and GFP-positive hESC clones obtained through co-transfection of pattP4X-PGKssPuro-UTF1-eGFP and pEF1α-ss-Int-C3CNLS . ( C ) Southern blot analysis. Genomic DNA from three hESC clones and parental hESC cells were digested with NdeI or XbaI. Digoxigenin-labeled probe to EGFP was employed. Lanes: M1, 1 kb DNA ladder; 10 8 , 10 9 , copies of linearized targeting vector as positive control; ES, parental DNA; A3, E3 and K3, genomic DNA from targeted hESC clones. White arrow heads indicate fragments of the expected size and black arrow heads indicate extra or unexpected fragments in the targeted clones. ( D ) Functional test for UTF1 promoter-driven EGFP expression in targeted hESC clones. Fluorescence microscopic analysis of undifferentiated and RA-induced, differentiated parental hES-047 cells and clones A3, E3 and K3. EGFP expression was detected with the undifferentiated hESC clones A3, E3 and K3 (column 2, panels 2, 3 and 4) but disappeared in differentiated progenies (column 4, panels 2, 3 and 4) respectively. Panels in columns 1 and 3 are phase-contrast light micrographs of undifferentiated and differentiated cells, respectively. Magnification 5×; Scale bars 100 μm. ( E ) FACS analysis for undifferentiated and differentiated hESCs clones Dot plots representing GFP + cells (upper right quadrant) and GFP − cells (lower right quadrant) for the untargeted hESCs, undifferentiated targeted hESC clones (A3, E3, K3) after 3 weeks (early) and 8 weeks (late) of culturing the cells (left and middle panel) and their differentiated progenies (right panel).

    Techniques Used: Plasmid Preparation, Clone Assay, Nested PCR, Polymerase Chain Reaction, Negative Control, Cotransfection, Southern Blot, Labeling, Positive Control, Functional Assay, Expressing, Fluorescence, FACS

    att H4X targeting in human embryonic stem cell (hESCs). ( A ) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into att H4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. ( B ) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS ( pCMVssInt-C3C ), 6xHIS-tagged Int-C3CNLS ( pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H ) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). ( C ) Example of screening for att H4X × att P4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_ att H4X_F2 and att P rev (for the left junction; top left panel) and cs_ att H4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers att P rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (10 7 , 10 8 ), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP . White arrow heads indicate fragments of the expected size in the targeted clones.
    Figure Legend Snippet: att H4X targeting in human embryonic stem cell (hESCs). ( A ) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into att H4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. ( B ) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS ( pCMVssInt-C3C ), 6xHIS-tagged Int-C3CNLS ( pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H ) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). ( C ) Example of screening for att H4X × att P4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_ att H4X_F2 and att P rev (for the left junction; top left panel) and cs_ att H4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers att P rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (10 7 , 10 8 ), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP . White arrow heads indicate fragments of the expected size in the targeted clones.

    Techniques Used: Plasmid Preparation, Western Blot, Expressing, Transfection, Purification, Positive Control, Polymerase Chain Reaction, Amplification, Sequencing, Negative Control, Clone Assay, Cotransfection, Southern Blot

    Off-target analysis in NEB-1 and HeLa cell lines. ( A ) Schematic drawing showing self-ligation of PstI digested genomic DNA consisting of integrated pattP4X-PGKssPuro target vector. Positions of relevant primers (Puro rev24, Puro rev303, pr21, attP fwd) used for inverse nested PCR to determine the potential att R site are indicated. ( B ) Screening for off-targeting events in NEB-1 clones. Inverse nested PCR was performed using genomic DNA from 26 puromycin-resistant NEB-1 clones which were obtained from two independent transfections. PCR products that were not present in control PCRs (with genomic DNA of NEB-1 parental cells as template) and were larger than 200 bp (fragments marked with black arrows) were detected in clones N1-C7, N1-C14, N2-C1, N2-C2, N2-C3, N2-C4, N2-C6, N2-C10, N2-C11 (upper two panels). They were extracted and re-amplified (bottom panel) for sequencing. W, no DNA template control; NEB, negative control (genomic DNA from parental cells); Bulk 1 bulk 2, genomic DNA template from all puromycin resistant colonies obtained through two independent co-transfections of pattP4X-PGKssPuro and pCMVssInt-h/218CNL; N1(C1-C14) and N2 (C1-C12), genomic DNA from puromycin resistant NEB-1 clones obtained through two independent co-transfections of pattP4X-PGKssPuro and pCMVssInt-C3CNLS ; +, positive control (genomic DNA from a NEB-1 clone carrying an att H4X × att P4X integration event); M, 100 bp DNA ladder; M1, 1 kb DNA ladder. ( C ) Table showing sequence analysis of NEB-1 targeted clones (nature, possible mechanism and chromosomal location of genomic integration of target vector). ( D ) Table showing the observed off-target sequences based on att P4X targeting in NEB-1 and HeLa cell lines. ( E ) Sequence logo analysis for the 21bp off-target att H (HOH‘) sequences found based on att P4X targeting in NEB-1 and HeLa cell lines.
    Figure Legend Snippet: Off-target analysis in NEB-1 and HeLa cell lines. ( A ) Schematic drawing showing self-ligation of PstI digested genomic DNA consisting of integrated pattP4X-PGKssPuro target vector. Positions of relevant primers (Puro rev24, Puro rev303, pr21, attP fwd) used for inverse nested PCR to determine the potential att R site are indicated. ( B ) Screening for off-targeting events in NEB-1 clones. Inverse nested PCR was performed using genomic DNA from 26 puromycin-resistant NEB-1 clones which were obtained from two independent transfections. PCR products that were not present in control PCRs (with genomic DNA of NEB-1 parental cells as template) and were larger than 200 bp (fragments marked with black arrows) were detected in clones N1-C7, N1-C14, N2-C1, N2-C2, N2-C3, N2-C4, N2-C6, N2-C10, N2-C11 (upper two panels). They were extracted and re-amplified (bottom panel) for sequencing. W, no DNA template control; NEB, negative control (genomic DNA from parental cells); Bulk 1 bulk 2, genomic DNA template from all puromycin resistant colonies obtained through two independent co-transfections of pattP4X-PGKssPuro and pCMVssInt-h/218CNL; N1(C1-C14) and N2 (C1-C12), genomic DNA from puromycin resistant NEB-1 clones obtained through two independent co-transfections of pattP4X-PGKssPuro and pCMVssInt-C3CNLS ; +, positive control (genomic DNA from a NEB-1 clone carrying an att H4X × att P4X integration event); M, 100 bp DNA ladder; M1, 1 kb DNA ladder. ( C ) Table showing sequence analysis of NEB-1 targeted clones (nature, possible mechanism and chromosomal location of genomic integration of target vector). ( D ) Table showing the observed off-target sequences based on att P4X targeting in NEB-1 and HeLa cell lines. ( E ) Sequence logo analysis for the 21bp off-target att H (HOH‘) sequences found based on att P4X targeting in NEB-1 and HeLa cell lines.

    Techniques Used: Ligation, Plasmid Preparation, Nested PCR, Clone Assay, Transfection, Polymerase Chain Reaction, Amplification, Sequencing, Negative Control, Positive Control

    37) Product Images from "Expression and Activity of a Novel Cathelicidin from Domestic Cats"

    Article Title: Expression and Activity of a Novel Cathelicidin from Domestic Cats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018756

    3′ RACE Analysis and Identification of feCath. (A) Schematic of 3′ RACE strategy targeting the signal sequence region and the propeptide (cathelin) domain with sense primers and using antisense adaptor primer, AP1. Feline bone marrow RNA was reverse transcribed with oligo-dT conjugated to adaptor primer 1/2 (AP1/2). Sense primers (sequences found in Table 1 ) were used to amplify cathelicidin related sequences in the pool of bone marrow cDNA. (B) Agarose gel electrophoresis analysis of 3′ RACE PCR products from (A). HaeIII digested Phi-X174 phage DNA used as the marker.
    Figure Legend Snippet: 3′ RACE Analysis and Identification of feCath. (A) Schematic of 3′ RACE strategy targeting the signal sequence region and the propeptide (cathelin) domain with sense primers and using antisense adaptor primer, AP1. Feline bone marrow RNA was reverse transcribed with oligo-dT conjugated to adaptor primer 1/2 (AP1/2). Sense primers (sequences found in Table 1 ) were used to amplify cathelicidin related sequences in the pool of bone marrow cDNA. (B) Agarose gel electrophoresis analysis of 3′ RACE PCR products from (A). HaeIII digested Phi-X174 phage DNA used as the marker.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    38) Product Images from "Dysregulated immune system networks in war veterans with PTSD is an outcome of altered miRNA expression and DNA methylation"

    Article Title: Dysregulated immune system networks in war veterans with PTSD is an outcome of altered miRNA expression and DNA methylation

    Journal: Scientific Reports

    doi: 10.1038/srep31209

    DNA methylation level has a trend that corroborates gene expression. There is a clear trend showing higher DNA methylation and lowered mRNA levels and vice-versa for the corresponding gene. (a) DNA methylation levels of the select genes presented as box plot. On x-axis, the names of gene are provided and y-axis provides the average β- values of DNA methylation. The two bars corresponding to each gene represent the DNA methylation level for control followed by PTSD patient in a left to right direction. (b) Transcript levels (y-axis: log 2 fold change values) of genes, after RNA-Seq analysis, listed in Fig. 4a. (c) Real time PCR validation of differentially expressed genes. To validate the RNA-Seq results, qRT-PCR was performed for seven representative genes with cDNA prepared from total RNA obtained from PBMCs of 24 control and 24 PTSD patients. The values are relative abundance (RA) values after qRT-PCR. The table inside the figure provides log 2 fold change values of the respective genes after RNA-Seq analysis. The error bars indicate standard error.
    Figure Legend Snippet: DNA methylation level has a trend that corroborates gene expression. There is a clear trend showing higher DNA methylation and lowered mRNA levels and vice-versa for the corresponding gene. (a) DNA methylation levels of the select genes presented as box plot. On x-axis, the names of gene are provided and y-axis provides the average β- values of DNA methylation. The two bars corresponding to each gene represent the DNA methylation level for control followed by PTSD patient in a left to right direction. (b) Transcript levels (y-axis: log 2 fold change values) of genes, after RNA-Seq analysis, listed in Fig. 4a. (c) Real time PCR validation of differentially expressed genes. To validate the RNA-Seq results, qRT-PCR was performed for seven representative genes with cDNA prepared from total RNA obtained from PBMCs of 24 control and 24 PTSD patients. The values are relative abundance (RA) values after qRT-PCR. The table inside the figure provides log 2 fold change values of the respective genes after RNA-Seq analysis. The error bars indicate standard error.

    Techniques Used: DNA Methylation Assay, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    39) Product Images from "A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine"

    Article Title: A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042732

    Circulating HAdV types in southern Palestine. HAdV types circulating in Southern Palestine from 2005 to 2010 were detected using nested PCR and DNA sequencing assay of the HAdV hexon gene HVR1–6 followed by sequencing and sequence analysis. The 44 sequences were assigned the GenBank accession numbers [JQ796022–JQ796065]. The percentage of each serotype was calculated relatively to the number of total sequenced samples (44 samples).
    Figure Legend Snippet: Circulating HAdV types in southern Palestine. HAdV types circulating in Southern Palestine from 2005 to 2010 were detected using nested PCR and DNA sequencing assay of the HAdV hexon gene HVR1–6 followed by sequencing and sequence analysis. The 44 sequences were assigned the GenBank accession numbers [JQ796022–JQ796065]. The percentage of each serotype was calculated relatively to the number of total sequenced samples (44 samples).

    Techniques Used: Nested PCR, DNA Sequencing, Sequencing

    40) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

    Related Articles

    Clone Assay:

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression
    Article Snippet: .. For sequencing (GATC, Konstanz, Germany), the LAM-PCR amplicons were purified (PCR purification kit; Qiagen) and shotgun cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA). ..

    Laser Capture Microdissection:

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression
    Article Snippet: .. For sequencing (GATC, Konstanz, Germany), the LAM-PCR amplicons were purified (PCR purification kit; Qiagen) and shotgun cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA). ..

    Purification:

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase
    Article Snippet: .. DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA). .. Most chemicals and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO). pSV-β-Gal vector containing lacZ was purchased from Promega (Madison, WI).

    Article Title: Rapid one-step recombinational cloning
    Article Snippet: .. DNA and PCR product purification kits were purchased from Qiagen (Germany). .. Gateway® LR Clonase mix was purchased from Invitrogen.

    Article Title: Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension
    Article Snippet: .. Chromatin was treated with RNase A, proteinase K, and the DNA was column purified (PCR Purification kit, Qiagen). .. Purified DNA was PCR amplified using primers ( ) targeting the Anf , Renin and Apob promoter regions, and Hbb used as a negative control.

    Article Title: Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens
    Article Snippet: .. Plasmid DNA purification was carried out using Mini plasmid purification kits and PCR purification kits (QIAGEN, Inc., Valencia, Calif.). .. The deletion mutants Δ omcT :: spec , ΔomcS :: spec , and Δ omcE :: kan were transformed with pMJG- omcS , pMJG- omcT , and pMJG- omcE , respectively, as previously described ( ).

    Article Title: HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin
    Article Snippet: .. Finally, samples were treated with 20μg Proteinase K (Invitrogen) for 1 hour at 37 °C, and DNA was purified using QIAGEN PCR purification kits and spin columns. .. Microarray analysis of MNase–ChIP and ChIP experiments MNase–ChIP and ChIP samples were competitively hybridized with their respective input samples.

    Article Title: Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1
    Article Snippet: .. After incubation for 1 hr at 37°C, ChIP DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. .. Quantitative Real-time PCR Analysis Real-time PCR was conducted using SYBR Premix EX Taq (Takara) with LightCycler480 (Roche Diagnostics).

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression
    Article Snippet: .. For sequencing (GATC, Konstanz, Germany), the LAM-PCR amplicons were purified (PCR purification kit; Qiagen) and shotgun cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1
    Article Snippet: .. After incubation for 1 hr at 37°C, ChIP DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. .. Quantitative Real-time PCR Analysis Real-time PCR was conducted using SYBR Premix EX Taq (Takara) with LightCycler480 (Roche Diagnostics).

    Polymerase Chain Reaction:

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase
    Article Snippet: .. DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA). .. Most chemicals and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO). pSV-β-Gal vector containing lacZ was purchased from Promega (Madison, WI).

    Article Title: Rapid one-step recombinational cloning
    Article Snippet: .. DNA and PCR product purification kits were purchased from Qiagen (Germany). .. Gateway® LR Clonase mix was purchased from Invitrogen.

    Article Title: Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension
    Article Snippet: .. Chromatin was treated with RNase A, proteinase K, and the DNA was column purified (PCR Purification kit, Qiagen). .. Purified DNA was PCR amplified using primers ( ) targeting the Anf , Renin and Apob promoter regions, and Hbb used as a negative control.

    Article Title: Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens
    Article Snippet: .. Plasmid DNA purification was carried out using Mini plasmid purification kits and PCR purification kits (QIAGEN, Inc., Valencia, Calif.). .. The deletion mutants Δ omcT :: spec , ΔomcS :: spec , and Δ omcE :: kan were transformed with pMJG- omcS , pMJG- omcT , and pMJG- omcE , respectively, as previously described ( ).

    Article Title: HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin
    Article Snippet: .. Finally, samples were treated with 20μg Proteinase K (Invitrogen) for 1 hour at 37 °C, and DNA was purified using QIAGEN PCR purification kits and spin columns. .. Microarray analysis of MNase–ChIP and ChIP experiments MNase–ChIP and ChIP samples were competitively hybridized with their respective input samples.

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression
    Article Snippet: .. For sequencing (GATC, Konstanz, Germany), the LAM-PCR amplicons were purified (PCR purification kit; Qiagen) and shotgun cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA). ..

    Incubation:

    Article Title: Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1
    Article Snippet: .. After incubation for 1 hr at 37°C, ChIP DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. .. Quantitative Real-time PCR Analysis Real-time PCR was conducted using SYBR Premix EX Taq (Takara) with LightCycler480 (Roche Diagnostics).

    DNA Purification:

    Article Title: Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens
    Article Snippet: .. Plasmid DNA purification was carried out using Mini plasmid purification kits and PCR purification kits (QIAGEN, Inc., Valencia, Calif.). .. The deletion mutants Δ omcT :: spec , ΔomcS :: spec , and Δ omcE :: kan were transformed with pMJG- omcS , pMJG- omcT , and pMJG- omcE , respectively, as previously described ( ).

    Sequencing:

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression
    Article Snippet: .. For sequencing (GATC, Konstanz, Germany), the LAM-PCR amplicons were purified (PCR purification kit; Qiagen) and shotgun cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA). ..

    Chromatin Immunoprecipitation:

    Article Title: Regulation of DNA Replication Timing on Human Chromosome by a Cell-Type Specific DNA Binding Protein SATB1
    Article Snippet: .. After incubation for 1 hr at 37°C, ChIP DNA was purified by MinElute (QIAGEN) and used for quantitative PCR. .. Quantitative Real-time PCR Analysis Real-time PCR was conducted using SYBR Premix EX Taq (Takara) with LightCycler480 (Roche Diagnostics).

    Plasmid Preparation:

    Article Title: Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens
    Article Snippet: .. Plasmid DNA purification was carried out using Mini plasmid purification kits and PCR purification kits (QIAGEN, Inc., Valencia, Calif.). .. The deletion mutants Δ omcT :: spec , ΔomcS :: spec , and Δ omcE :: kan were transformed with pMJG- omcS , pMJG- omcT , and pMJG- omcE , respectively, as previously described ( ).

    Article Title: Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression
    Article Snippet: .. For sequencing (GATC, Konstanz, Germany), the LAM-PCR amplicons were purified (PCR purification kit; Qiagen) and shotgun cloned into the TOPO TA vector (Invitrogen, Carlsbad, CA). ..

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    Qiagen pcr product purification kits
    Single fragment cloning by Hot Fusion. (A) A diagram of single fragment cloning in a binary base vector. Base vector containing a lacZ gene was linearized by restriction enzyme digestion with AscI and KpnI to release lacZ . The linearized vector was directly used for Hot Fusion. (B) Amplified <t>PCR</t> products (A1 through A2) of plant gene promoters were used for cloning. M is a NEB 1 kb <t>DNA</t> ladder. (C) Transformation plates of cloned PCR products (A1 and H1 are not shown). Blue colonies on the plates contain the parental vector and white colonies contain the potential recombinants. Eight white colonies were screened for each construct ( Table 2 ).
    Pcr Product Purification Kits, supplied by Qiagen, used in various techniques. Bioz Stars score: 97/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Single fragment cloning by Hot Fusion. (A) A diagram of single fragment cloning in a binary base vector. Base vector containing a lacZ gene was linearized by restriction enzyme digestion with AscI and KpnI to release lacZ . The linearized vector was directly used for Hot Fusion. (B) Amplified PCR products (A1 through A2) of plant gene promoters were used for cloning. M is a NEB 1 kb DNA ladder. (C) Transformation plates of cloned PCR products (A1 and H1 are not shown). Blue colonies on the plates contain the parental vector and white colonies contain the potential recombinants. Eight white colonies were screened for each construct ( Table 2 ).

    Journal: PLoS ONE

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    doi: 10.1371/journal.pone.0115318

    Figure Lengend Snippet: Single fragment cloning by Hot Fusion. (A) A diagram of single fragment cloning in a binary base vector. Base vector containing a lacZ gene was linearized by restriction enzyme digestion with AscI and KpnI to release lacZ . The linearized vector was directly used for Hot Fusion. (B) Amplified PCR products (A1 through A2) of plant gene promoters were used for cloning. M is a NEB 1 kb DNA ladder. (C) Transformation plates of cloned PCR products (A1 and H1 are not shown). Blue colonies on the plates contain the parental vector and white colonies contain the potential recombinants. Eight white colonies were screened for each construct ( Table 2 ).

    Article Snippet: DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA).

    Techniques: Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Transformation Assay, Construct

    PCR screening of multi-fragment assembled clones from Table 3 . Eight clones (colonies) from each construct were grown overnight in the 96-well plate containing LB + Spectinomycin (50 ug/ml). One µl of the over-night grown cell culture was used for PCR screening with base vector primers (18 bp) located immediately upstream of the promoter and downstream of the terminator. PCR products for each construct were loaded every other lane on a 1.0% agarose gel (containing ethidium bromide) with a multi channel pipette. GeneRuler 1 kb Plus DNA Ladder was used as the DNA marker.

    Journal: PLoS ONE

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    doi: 10.1371/journal.pone.0115318

    Figure Lengend Snippet: PCR screening of multi-fragment assembled clones from Table 3 . Eight clones (colonies) from each construct were grown overnight in the 96-well plate containing LB + Spectinomycin (50 ug/ml). One µl of the over-night grown cell culture was used for PCR screening with base vector primers (18 bp) located immediately upstream of the promoter and downstream of the terminator. PCR products for each construct were loaded every other lane on a 1.0% agarose gel (containing ethidium bromide) with a multi channel pipette. GeneRuler 1 kb Plus DNA Ladder was used as the DNA marker.

    Article Snippet: DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA).

    Techniques: Polymerase Chain Reaction, Clone Assay, Construct, Cell Culture, Plasmid Preparation, Agarose Gel Electrophoresis, Transferring, Marker

    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Journal: PLoS ONE

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    doi: 10.1371/journal.pone.0115318

    Figure Lengend Snippet: A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Article Snippet: DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Generated, Transformation Assay

    PCR confirmation of genes cloned into Kan-resistant vector (top panel) and Spec-resistant vector (bottom panel). Both cultured cells and plasmid DNAs were used for PCR confirmation. The 1 kb DNA marker (New England Biolabs) was used between every16 lanes. Samples were loaded using a multi-channel pipette, which loads every other well resulting in an interleaved loading pattern. In the upper panel, four individual colonies were screened for each clone using universal primers, which anneal to the vector sequences. Accordingly, clones without the insert amplify a small band of 200 bp. In the bottom panel, eight colonies from each clone were screened using gene-specific primers, so no band is observed for clones that do not contain the desired insert. While the use of gene-specific primers is useful for confirming the identity of a given clone, false negatives can arise due to PCR failures.

    Journal: Nucleic Acids Research

    Article Title: Rapid one-step recombinational cloning

    doi: 10.1093/nar/gkn167

    Figure Lengend Snippet: PCR confirmation of genes cloned into Kan-resistant vector (top panel) and Spec-resistant vector (bottom panel). Both cultured cells and plasmid DNAs were used for PCR confirmation. The 1 kb DNA marker (New England Biolabs) was used between every16 lanes. Samples were loaded using a multi-channel pipette, which loads every other well resulting in an interleaved loading pattern. In the upper panel, four individual colonies were screened for each clone using universal primers, which anneal to the vector sequences. Accordingly, clones without the insert amplify a small band of 200 bp. In the bottom panel, eight colonies from each clone were screened using gene-specific primers, so no band is observed for clones that do not contain the desired insert. While the use of gene-specific primers is useful for confirming the identity of a given clone, false negatives can arise due to PCR failures.

    Article Snippet: DNA and PCR product purification kits were purchased from Qiagen (Germany).

    Techniques: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Cell Culture, Marker, Transferring

    Comparison of the PCR results using truncated attL sites and different DNA polymerases. HiFi tag refers to high-fidelity tag polymerase. Hercu refers to herculase. Pfu refers to Pfu turbo polymerases.

    Journal: Nucleic Acids Research

    Article Title: Rapid one-step recombinational cloning

    doi: 10.1093/nar/gkn167

    Figure Lengend Snippet: Comparison of the PCR results using truncated attL sites and different DNA polymerases. HiFi tag refers to high-fidelity tag polymerase. Hercu refers to herculase. Pfu refers to Pfu turbo polymerases.

    Article Snippet: DNA and PCR product purification kits were purchased from Qiagen (Germany).

    Techniques: Polymerase Chain Reaction

    Expression of omcS , omcT , and omcE . (A) The expression of omcS , omcT , and omcE was measured by RT-PCR. RNAs were extracted from wild-type cells grown in the presence of fumarate (F), ferric citrate (Fe sol ), and insoluble Fe(III) oxide (Fe insol ). Negative (−) controls were performed without cDNA synthesis, and positive controls (+) were performed by using genomic DNA as the template. (B) Northern analysis of omcS and omcT expression in cells grown on fumarate. The expression of omcS in the wild type (lane 1) and the Δ omcS :: spec (lane 2) and Δ omcT :: spec (lane 3) mutants is shown. The expression of omcT in the wild type (lane 4) and the Δ omcS :: spec (lane 5) and Δ omcT :: spec (lane 6) mutants is also indicated. (C) Expression of omcS and omcT in deletion mutants. The strains were cultured with fumarate as the electron acceptor, and genomic DNA was used as a template for positive controls. MW, molecular size marker.

    Journal: Applied and Environmental Microbiology

    Article Title: Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens

    doi: 10.1128/AEM.71.12.8634-8641.2005

    Figure Lengend Snippet: Expression of omcS , omcT , and omcE . (A) The expression of omcS , omcT , and omcE was measured by RT-PCR. RNAs were extracted from wild-type cells grown in the presence of fumarate (F), ferric citrate (Fe sol ), and insoluble Fe(III) oxide (Fe insol ). Negative (−) controls were performed without cDNA synthesis, and positive controls (+) were performed by using genomic DNA as the template. (B) Northern analysis of omcS and omcT expression in cells grown on fumarate. The expression of omcS in the wild type (lane 1) and the Δ omcS :: spec (lane 2) and Δ omcT :: spec (lane 3) mutants is shown. The expression of omcT in the wild type (lane 4) and the Δ omcS :: spec (lane 5) and Δ omcT :: spec (lane 6) mutants is also indicated. (C) Expression of omcS and omcT in deletion mutants. The strains were cultured with fumarate as the electron acceptor, and genomic DNA was used as a template for positive controls. MW, molecular size marker.

    Article Snippet: Plasmid DNA purification was carried out using Mini plasmid purification kits and PCR purification kits (QIAGEN, Inc., Valencia, Calif.).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Cell Culture, Marker

    Increased H3 turnover in heterochromatin mutants is not solely due to changes in RNAPII transcription ( a ) Histone H3 replacement (blue) was measured across the mating type locus using MNase–ChIP–on–Chip analysis as described in Fig 1 . RNAPII occupancy (ChIP vs Input) was measured by ChIP–on–Chip in clr3 Δ, clr4 Δ or wild–type (WT) cells and plotted in alignment with the map (red). ( b ) RT–PCR analysis performed using total RNA samples isolated from clr3 Δ, clr4 Δ or wild–type (WT) cells. Genomic DNA (gDNA) was used as a control. The locations amplified by primer pairs 49, 51, 65 and 70 are highlighted with red shading (see online methods for primer references). Heterochromatin and euchromatin portions of the mating–type region are indicated at the top.

    Journal: Nature structural & molecular biology

    Article Title: HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin

    doi: 10.1038/nsmb.2565

    Figure Lengend Snippet: Increased H3 turnover in heterochromatin mutants is not solely due to changes in RNAPII transcription ( a ) Histone H3 replacement (blue) was measured across the mating type locus using MNase–ChIP–on–Chip analysis as described in Fig 1 . RNAPII occupancy (ChIP vs Input) was measured by ChIP–on–Chip in clr3 Δ, clr4 Δ or wild–type (WT) cells and plotted in alignment with the map (red). ( b ) RT–PCR analysis performed using total RNA samples isolated from clr3 Δ, clr4 Δ or wild–type (WT) cells. Genomic DNA (gDNA) was used as a control. The locations amplified by primer pairs 49, 51, 65 and 70 are highlighted with red shading (see online methods for primer references). Heterochromatin and euchromatin portions of the mating–type region are indicated at the top.

    Article Snippet: Finally, samples were treated with 20μg Proteinase K (Invitrogen) for 1 hour at 37 °C, and DNA was purified using QIAGEN PCR purification kits and spin columns.

    Techniques: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification

    Clr3–dependent suppression of histone turnover correlates with epigenetic stability of heterochromatin ( a ) ChIP analysis of Clr3 localization of at the silent mat region. Strains expressing Myc tagged Clr3 in K Δ ∷ura4 + ura4–on or ura4–off state were used to perform ChIP. ChIP DNA was analyzed by semi–quantitative competitive PCR using primers that amplify both full–length K Δ ∷ura4 + and endogenous mini– ura4 ( ura4DSE ) as internal control. The relative enrichments were determined by calculating the ratio of the band intensities of [ChIP K Δ ∷ura4 + ÷ ChIP ura4DSE ] ÷ [Input K Δ ∷ura4 + ÷ Input ura4DSE ]. Results were confirmed by quantitative real–time PCR (qPCR). Relative enrichment of K Δ ∷ura4 + was normalized against untagged negative control and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 3 independent biological replicates (n=3) ( b ) H3 replacement was measured in K Δ ∷ura4 + ura4–on or ura4–off cells. The endogenous ura4 + was deleted in the strains used. ( c ) ChIP analysis of H3K9me2 levels at K Δ ∷ura4 + ura4–on cells. Experiments were performed with the same strains used in a . H3K9me levels were confirmed by qPCR and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 4 independent biological replicates (n=4).

    Journal: Nature structural & molecular biology

    Article Title: HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin

    doi: 10.1038/nsmb.2565

    Figure Lengend Snippet: Clr3–dependent suppression of histone turnover correlates with epigenetic stability of heterochromatin ( a ) ChIP analysis of Clr3 localization of at the silent mat region. Strains expressing Myc tagged Clr3 in K Δ ∷ura4 + ura4–on or ura4–off state were used to perform ChIP. ChIP DNA was analyzed by semi–quantitative competitive PCR using primers that amplify both full–length K Δ ∷ura4 + and endogenous mini– ura4 ( ura4DSE ) as internal control. The relative enrichments were determined by calculating the ratio of the band intensities of [ChIP K Δ ∷ura4 + ÷ ChIP ura4DSE ] ÷ [Input K Δ ∷ura4 + ÷ Input ura4DSE ]. Results were confirmed by quantitative real–time PCR (qPCR). Relative enrichment of K Δ ∷ura4 + was normalized against untagged negative control and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 3 independent biological replicates (n=3) ( b ) H3 replacement was measured in K Δ ∷ura4 + ura4–on or ura4–off cells. The endogenous ura4 + was deleted in the strains used. ( c ) ChIP analysis of H3K9me2 levels at K Δ ∷ura4 + ura4–on cells. Experiments were performed with the same strains used in a . H3K9me levels were confirmed by qPCR and the mean enrichment is presented. Error bars represent standard error of the mean calculated from 4 independent biological replicates (n=4).

    Article Snippet: Finally, samples were treated with 20μg Proteinase K (Invitrogen) for 1 hour at 37 °C, and DNA was purified using QIAGEN PCR purification kits and spin columns.

    Techniques: Chromatin Immunoprecipitation, Expressing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Negative Control