ampin1c  (Qiagen)

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

    Qiagen ampin1c
    QIAquick PCR Purification Kit
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    https://www.bioz.com/result/ampin1c/product/Qiagen
    Average 91 stars, based on 31295 article reviews
    Price from $9.99 to $1999.99
    ampin1c - by Bioz Stars, 2020-08
    91/100 stars

    Images

    1) Product Images from "Generation of shape complexity through tissue conflict resolution"

    Article Title: Generation of shape complexity through tissue conflict resolution

    Journal: eLife

    doi: 10.7554/eLife.20156

    Expression patterns of AmPIN1 genes. ( A-L ) Expression patterns of AmPIN1 genes at 8 DAI ( A-C ), 10 DAI ( D-F ), 12 DAI ( G-I ) and 14 DAI ( J-L ), determined by RNA in situ hybridisation of alternative sections hybridised with AmPIN1a (left panels, A , D , G , J ), AmPIN1b (middle panels, B , E , H , K ) and AmPIN1c (right panels, C , F , I , L ) probes. The AmPIN1 genes show similar expression patterns in the vascular tissue of petal primordia ( A-F ), with some localised differences particularly at the tips of the petal primordia ( D-F and close-ups of red boxes in respective D-F images), in particular for AmPIN1c which shows stronger epidermal expression at the petal tips. At 12 DAI, the AmPIN1 genes continue to be expressed in the vascular tissue ( G-I ) but are additionally detected at the epidermis of the palate and lip regions, particularly strongly for AmPIN1c (white arrows in close-ups of blue boxes in respective G-I images). At 13 DAI, the epidermal Am PIN1 expression starts to decrease ( J-L ) in comparison with the vascular tissue signal which continues to be similar to previous stages (close-ups of green squares in respective J-L images). The upper and lower limits of the palate and lip domains are marked by pink dashed lines. The diagram in the upper right corner of every left panel represents the orientation of the section (white line) relative to the five petals (red-dorsal, orange -lateral and yellow -ventral). V: ventral petal; D: dorsal petal; st: stamen; ca: carpel. Scale bars, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.20156.028
    Figure Legend Snippet: Expression patterns of AmPIN1 genes. ( A-L ) Expression patterns of AmPIN1 genes at 8 DAI ( A-C ), 10 DAI ( D-F ), 12 DAI ( G-I ) and 14 DAI ( J-L ), determined by RNA in situ hybridisation of alternative sections hybridised with AmPIN1a (left panels, A , D , G , J ), AmPIN1b (middle panels, B , E , H , K ) and AmPIN1c (right panels, C , F , I , L ) probes. The AmPIN1 genes show similar expression patterns in the vascular tissue of petal primordia ( A-F ), with some localised differences particularly at the tips of the petal primordia ( D-F and close-ups of red boxes in respective D-F images), in particular for AmPIN1c which shows stronger epidermal expression at the petal tips. At 12 DAI, the AmPIN1 genes continue to be expressed in the vascular tissue ( G-I ) but are additionally detected at the epidermis of the palate and lip regions, particularly strongly for AmPIN1c (white arrows in close-ups of blue boxes in respective G-I images). At 13 DAI, the epidermal Am PIN1 expression starts to decrease ( J-L ) in comparison with the vascular tissue signal which continues to be similar to previous stages (close-ups of green squares in respective J-L images). The upper and lower limits of the palate and lip domains are marked by pink dashed lines. The diagram in the upper right corner of every left panel represents the orientation of the section (white line) relative to the five petals (red-dorsal, orange -lateral and yellow -ventral). V: ventral petal; D: dorsal petal; st: stamen; ca: carpel. Scale bars, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.20156.028

    Techniques Used: Expressing, In Situ, Hybridization

    2) Product Images from "Targeted mutagenesis in a human-parasitic nematode"

    Article Title: Targeted mutagenesis in a human-parasitic nematode

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006675

    CRISPR-mediated mutagenesis of Ss-unc-22 results in putative deletion of the target locus. ( A ) Representative gel of wild-type iL3s (top) or unc F 1 iL3s from RNP injections at site #3 (bottom). Genomic DNA from each iL3 was split into two reactions: ctrl. = control reaction amplifying 416 bp of the first exon of the Ss-act-2 gene to confirm the presence of genomic DNA; u22 = reaction amplifying 660 bp around site #3. Size markers = 1.5 kb, 1 kb, and 500 bp from top to bottom. ( B ) The Ss-unc-22 region is significantly depleted in unc F 1 iL3s. Left: relative quantity analysis of PCR products. All control bands and all u22 bands were quantified relative to their respective reference bands, denoted by asterisks in A . Values > 1 indicate more PCR product than the reference while values
    Figure Legend Snippet: CRISPR-mediated mutagenesis of Ss-unc-22 results in putative deletion of the target locus. ( A ) Representative gel of wild-type iL3s (top) or unc F 1 iL3s from RNP injections at site #3 (bottom). Genomic DNA from each iL3 was split into two reactions: ctrl. = control reaction amplifying 416 bp of the first exon of the Ss-act-2 gene to confirm the presence of genomic DNA; u22 = reaction amplifying 660 bp around site #3. Size markers = 1.5 kb, 1 kb, and 500 bp from top to bottom. ( B ) The Ss-unc-22 region is significantly depleted in unc F 1 iL3s. Left: relative quantity analysis of PCR products. All control bands and all u22 bands were quantified relative to their respective reference bands, denoted by asterisks in A . Values > 1 indicate more PCR product than the reference while values

    Techniques Used: CRISPR, Mutagenesis, Activated Clotting Time Assay, Polymerase Chain Reaction

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

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

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0247-4

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

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

    4) Product Images from "Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits"

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58586-3

    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Figure Legend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

    5) Product Images from "Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins"

    Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

    Journal: BMC Biology

    doi: 10.1186/1741-7007-2-21

    ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).
    Figure Legend Snippet: ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

    Techniques Used: Chromatin Immunoprecipitation, Modification, Methylation, Staining, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunoprecipitation

    6) Product Images from "Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins"

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148023

    Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.
    Figure Legend Snippet: Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Techniques Used: Sonication, Concentration Assay, Size-exclusion Chromatography, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated, Molecular Weight, Titration

    Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.
    Figure Legend Snippet: Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Techniques Used: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.
    Figure Legend Snippet: Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Techniques Used: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    7) Product Images from "RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples"

    Article Title: RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh116

    Hybridization of a set of standard liver cDNA mixtures containing increasing amounts of bacterial LysA antisense cDNA. ( A ) LysA abundance varying from approximately 9 to 9100 copies LysA cDNA per cell equivalent are shown in this figure (left vertical
    Figure Legend Snippet: Hybridization of a set of standard liver cDNA mixtures containing increasing amounts of bacterial LysA antisense cDNA. ( A ) LysA abundance varying from approximately 9 to 9100 copies LysA cDNA per cell equivalent are shown in this figure (left vertical

    Techniques Used: Hybridization

    8) Product Images from "MicroRNA-21 contributes to renal cell carcinoma cell invasiveness and angiogenesis via the PDCD4/c-Jun (AP-1) signalling pathway"

    Article Title: MicroRNA-21 contributes to renal cell carcinoma cell invasiveness and angiogenesis via the PDCD4/c-Jun (AP-1) signalling pathway

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2019.4928

    Transcription of miR-21 is activated by p-c-Jun. (A) Chromatin immunoprecipitation analysis demonstrated that p-c-Jun binds specifically to the pri-miR-21 promoter region. PCR amplification of the region containing the p-c-Jun recognition sequence in the pri-miR-21 DNA. (B) Inhibition of miR-21 expression by a decrease in c-Jun expression in A498 and 786-O cells. Transfection of A498 and 786-O cells with mock, NC or c-Jun siRNA for 24 h. c-Jun and miR-21 expression levels were examined using reverse transcription-quantitative PCR analysis and normalized to GAPDH and U6 snRNA expression, respectively. Data are presented as the mean ± standard error of the mean. ** P
    Figure Legend Snippet: Transcription of miR-21 is activated by p-c-Jun. (A) Chromatin immunoprecipitation analysis demonstrated that p-c-Jun binds specifically to the pri-miR-21 promoter region. PCR amplification of the region containing the p-c-Jun recognition sequence in the pri-miR-21 DNA. (B) Inhibition of miR-21 expression by a decrease in c-Jun expression in A498 and 786-O cells. Transfection of A498 and 786-O cells with mock, NC or c-Jun siRNA for 24 h. c-Jun and miR-21 expression levels were examined using reverse transcription-quantitative PCR analysis and normalized to GAPDH and U6 snRNA expression, respectively. Data are presented as the mean ± standard error of the mean. ** P

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Inhibition, Expressing, Transfection, Real-time Polymerase Chain Reaction

    9) Product Images from "Epigenetic Regulation of HYAL-1 Hyaluronidase Expression"

    Article Title: Epigenetic Regulation of HYAL-1 Hyaluronidase Expression

    Journal:

    doi: 10.1074/jbc.M801101200

    Analysis of HYAL-1 promoter methylation. Genomic DNA isolated from bladder and prostate cells was treated with sodium bisulfite and subjected to direct sequencing or methylation-specific real time PCR as described under “Experimental Procedures.”
    Figure Legend Snippet: Analysis of HYAL-1 promoter methylation. Genomic DNA isolated from bladder and prostate cells was treated with sodium bisulfite and subjected to direct sequencing or methylation-specific real time PCR as described under “Experimental Procedures.”

    Techniques Used: Methylation, Isolation, Sequencing, Real-time Polymerase Chain Reaction

    10) Product Images from "Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma"

    Article Title: Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma

    Journal: Nature Communications

    doi: 10.1038/ncomms14449

    Ets-1 activates the proximal NRAS promoter. ( a ) Immunoblot for FLAG in BRAF mutant SK-Mel29 cells (express low endogenous Usp9x and Ets-1 levels) stably transfected with FLAG-Usp9x or FLAG-Ets-1 (top). Relative luciferase units (firefly /Renilla ) in lysates from SK-Mel29 cells expressing (48 h) the proximal NRAS promoter, FLAG-Ets-1 or FLAG-Usp9x (bottom). ( b ) Immunoblot for FLAG in NRAS mutant WM1366 cells expressing FLAG-Ets-1 or FLAG-Usp9x (top). Relative luciferase units (firefly /Renilla ) in lysates from WM1366 cells expressing the proximal NRAS promoter, FLAG-Ets-1 or full-length FLAG-Usp9x (bottom). ( c ) Proximal NRAS promoter sequence cloned from NRAS mutant SK-Mel147 cells, highlighting 5 putative ETS sites (designated E1M through E5M) derived from ChIP-SEQ analysis in other cell lines and visual inspection of the sequence. The consensus ETS binding sequence is highlighted below (boxed). ( d ) Relative luciferase units (firefly /Renilla ) in lysates from SK-Mel29 cells expressing FLAG-Ets-1 and the proximal NRAS promoter (WT) or point mutants of each ETS putative binding site in the promoter region (E1M, E2M, E3M, E4M and E5M). ( e ) DNA-protein crosslinks from control and Usp9x KD cells were subjected to immunoprecipitation (as noted) before being used to prime a PCR reaction to detect the NRAS promoter. PCR products are shown (top) and compared with the input fraction (unfractionated DNA–protein complexes). Relative enrichment of the NRAS promoter for each condition is graphed below and represents the ave.±s.d. of three independent experiments.
    Figure Legend Snippet: Ets-1 activates the proximal NRAS promoter. ( a ) Immunoblot for FLAG in BRAF mutant SK-Mel29 cells (express low endogenous Usp9x and Ets-1 levels) stably transfected with FLAG-Usp9x or FLAG-Ets-1 (top). Relative luciferase units (firefly /Renilla ) in lysates from SK-Mel29 cells expressing (48 h) the proximal NRAS promoter, FLAG-Ets-1 or FLAG-Usp9x (bottom). ( b ) Immunoblot for FLAG in NRAS mutant WM1366 cells expressing FLAG-Ets-1 or FLAG-Usp9x (top). Relative luciferase units (firefly /Renilla ) in lysates from WM1366 cells expressing the proximal NRAS promoter, FLAG-Ets-1 or full-length FLAG-Usp9x (bottom). ( c ) Proximal NRAS promoter sequence cloned from NRAS mutant SK-Mel147 cells, highlighting 5 putative ETS sites (designated E1M through E5M) derived from ChIP-SEQ analysis in other cell lines and visual inspection of the sequence. The consensus ETS binding sequence is highlighted below (boxed). ( d ) Relative luciferase units (firefly /Renilla ) in lysates from SK-Mel29 cells expressing FLAG-Ets-1 and the proximal NRAS promoter (WT) or point mutants of each ETS putative binding site in the promoter region (E1M, E2M, E3M, E4M and E5M). ( e ) DNA-protein crosslinks from control and Usp9x KD cells were subjected to immunoprecipitation (as noted) before being used to prime a PCR reaction to detect the NRAS promoter. PCR products are shown (top) and compared with the input fraction (unfractionated DNA–protein complexes). Relative enrichment of the NRAS promoter for each condition is graphed below and represents the ave.±s.d. of three independent experiments.

    Techniques Used: Mutagenesis, Stable Transfection, Transfection, Luciferase, Expressing, Sequencing, Clone Assay, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    11) Product Images from "Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9"

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36736-y

    Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.
    Figure Legend Snippet: Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.

    Techniques Used: CRISPR, Plasmid Preparation, Software, Sequencing, Expressing, FACS, Transfection, Polymerase Chain Reaction, Clone Assay

    Generation of an all-in-one Cas9-mCherry-sgRNA vector using the Gateway system. ( A ) A Gateway entry plasmid was generated to clone a specific sgRNA into a PCR amplicon flanked by attL1 and attL2. PCR from the Entry vector using two sets of primers with specific 20 nt gRNA sequence (reaction 1 and 2) produces two fragments that partially overlap and can be combined by an overlap extension PCR (reaction 3). Multiplexing is possible by using multiple sets of Scaff-fwd and U6-Rev primers. ( B ) PCR products of two individual fragments and a full-length L1-U6-sgRNA-L2 are shown. ( C ) The final PCR amplicons can be combined with the adenoviral shuttle vector with R1-ccdB-R2 Destination sequence or used separately. pShuttle-Cas9-DEST was generated by cloning Cas9-mCherry and R1-R2 into pShuttle. ( D ) Gateway LR cloning produces 100% positive clones. Since the background (before recombination) clones cannot grow in ccdB-incompatible bacterial cell lines such as DH5a, all the transformed colonies with the LR reaction mixture contain the recombinant without ccdB, but with sgRNA. Two independent experiments are shown.
    Figure Legend Snippet: Generation of an all-in-one Cas9-mCherry-sgRNA vector using the Gateway system. ( A ) A Gateway entry plasmid was generated to clone a specific sgRNA into a PCR amplicon flanked by attL1 and attL2. PCR from the Entry vector using two sets of primers with specific 20 nt gRNA sequence (reaction 1 and 2) produces two fragments that partially overlap and can be combined by an overlap extension PCR (reaction 3). Multiplexing is possible by using multiple sets of Scaff-fwd and U6-Rev primers. ( B ) PCR products of two individual fragments and a full-length L1-U6-sgRNA-L2 are shown. ( C ) The final PCR amplicons can be combined with the adenoviral shuttle vector with R1-ccdB-R2 Destination sequence or used separately. pShuttle-Cas9-DEST was generated by cloning Cas9-mCherry and R1-R2 into pShuttle. ( D ) Gateway LR cloning produces 100% positive clones. Since the background (before recombination) clones cannot grow in ccdB-incompatible bacterial cell lines such as DH5a, all the transformed colonies with the LR reaction mixture contain the recombinant without ccdB, but with sgRNA. Two independent experiments are shown.

    Techniques Used: Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Sequencing, Multiplexing, Clone Assay, Transformation Assay, Recombinant

    12) Product Images from "Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis"

    Article Title: Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041996

    Representative profiles of the melting curves (derivative melt curves) of ITS-2 amplicons for Necator americanus (black), Ancylostoma duodenale (blue), A. ceylanicum (red), A. caninum (green) and A. braziliense (purple). N. americanus (black) produced two peaks while single peak was produced for other Ancylostoma spp. Pre-melt region: The set of lines to the left of the peak indicates the pre-melt start and stop temperatures when every amplicon is double-stranded. Post-melt region: The set of lines to the right of the peak indicates the post-melt start and stop temperatures when every amplicon is single-stranded.
    Figure Legend Snippet: Representative profiles of the melting curves (derivative melt curves) of ITS-2 amplicons for Necator americanus (black), Ancylostoma duodenale (blue), A. ceylanicum (red), A. caninum (green) and A. braziliense (purple). N. americanus (black) produced two peaks while single peak was produced for other Ancylostoma spp. Pre-melt region: The set of lines to the left of the peak indicates the pre-melt start and stop temperatures when every amplicon is double-stranded. Post-melt region: The set of lines to the right of the peak indicates the post-melt start and stop temperatures when every amplicon is single-stranded.

    Techniques Used: Produced, Amplification

    Representative profiles of the melting curves (difference plot curves) of ITS-2 amplicons for Necator americanus (black), Ancylostoma duodenale (blue), A. ceylanicum (red), A. caninum (green) and A. braziliense (purple).
    Figure Legend Snippet: Representative profiles of the melting curves (difference plot curves) of ITS-2 amplicons for Necator americanus (black), Ancylostoma duodenale (blue), A. ceylanicum (red), A. caninum (green) and A. braziliense (purple).

    Techniques Used:

    Representative profiles of the melting curves (aligned melt curves) of ITS-2 amplicons for Necator americanus (black), Ancylostoma duodenale (blue), A. ceylanicum (red), A. caninum (green) and A. braziliense (purple). Fluorescence is plotted against degrees Celsius (°C).
    Figure Legend Snippet: Representative profiles of the melting curves (aligned melt curves) of ITS-2 amplicons for Necator americanus (black), Ancylostoma duodenale (blue), A. ceylanicum (red), A. caninum (green) and A. braziliense (purple). Fluorescence is plotted against degrees Celsius (°C).

    Techniques Used: Fluorescence

    13) Product Images from "Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide"

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033287

    A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
    Figure Legend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

    Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.
    Figure Legend Snippet: Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction, Incubation, Immunoprecipitation, Transfection, Purification, Recombinant, Concentration Assay

    Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
    Figure Legend Snippet: Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Techniques Used: Expressing, Transfection, Isolation, Polymerase Chain Reaction, Activity Assay

    14) Product Images from "Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer"

    Article Title: Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039233

    Analysis of genomes isolated from rAAV5.DYSF. DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). “M” denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.
    Figure Legend Snippet: Analysis of genomes isolated from rAAV5.DYSF. DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). “M” denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.

    Techniques Used: Isolation, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction, Hybridization, Marker, Electron Microscopy

    Lack of truncated Dysferlin mRNA or protein in injected muscle. (A) RNA was extracted from injected tissue, converted to cDNA, and analyzed by PCR using 3 overlapping primer sets (Top to Bottom, 5′ to 3′ end) which showed that the entire Dysferlin transcript was amplified in injected muscle. Lane 1 – no template, lane 2 – injected muscle, lane 3 – injected muscle with no reverse transcriptase enzyme, lane 4 – uninjected muscle, lane 5 – human muscle control RNA, lane 6 – pAAV.MHCK7.DYSF control DNA. (B) Protein was extracted from injected tissue and analyzed by western blot. 4 mice were analyzed following injection with rAAV5.Dysf. A wild-type mouse muscle sample was used as a control. Both an N-terminal antibody and a C-terminal antibody were used for protein analysis in these muscles. Only the full-length Dysferlin band is unique to injected muscles.
    Figure Legend Snippet: Lack of truncated Dysferlin mRNA or protein in injected muscle. (A) RNA was extracted from injected tissue, converted to cDNA, and analyzed by PCR using 3 overlapping primer sets (Top to Bottom, 5′ to 3′ end) which showed that the entire Dysferlin transcript was amplified in injected muscle. Lane 1 – no template, lane 2 – injected muscle, lane 3 – injected muscle with no reverse transcriptase enzyme, lane 4 – uninjected muscle, lane 5 – human muscle control RNA, lane 6 – pAAV.MHCK7.DYSF control DNA. (B) Protein was extracted from injected tissue and analyzed by western blot. 4 mice were analyzed following injection with rAAV5.Dysf. A wild-type mouse muscle sample was used as a control. Both an N-terminal antibody and a C-terminal antibody were used for protein analysis in these muscles. Only the full-length Dysferlin band is unique to injected muscles.

    Techniques Used: Injection, Polymerase Chain Reaction, Amplification, Western Blot, Mouse Assay

    15) Product Images from "Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers"

    Article Title: Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers

    Journal: Current protocols in microbiology

    doi: 10.1002/9780471729259.mc01e04s20

    Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.
    Figure Legend Snippet: Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.

    Techniques Used: Ligation, Polymerase Chain Reaction

    Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis , and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.
    Figure Legend Snippet: Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis , and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.

    Techniques Used: Sequencing, Purification, Modification, Polymerase Chain Reaction

    16) Product Images from "Up-regulation and Sustained Activation of Stat1 Are Essential for Interferon-? (IFN-?)-induced Dual Oxidase 2 (Duox2) and Dual Oxidase A2 (DuoxA2) Expression in Human Pancreatic Cancer Cell Lines *"

    Article Title: Up-regulation and Sustained Activation of Stat1 Are Essential for Interferon-? (IFN-?)-induced Dual Oxidase 2 (Duox2) and Dual Oxidase A2 (DuoxA2) Expression in Human Pancreatic Cancer Cell Lines *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.191031

    An intact JAK-Stat1 signaling pathway and Stat1 up-regulation are essential for IFN-γ-induced Duox2 and DuoxA2 expression. A , signaling pathways involved in the regulation of Duox2 expression in BxPC-3 cells by IFN-γ. BxPC-3 cells were
    Figure Legend Snippet: An intact JAK-Stat1 signaling pathway and Stat1 up-regulation are essential for IFN-γ-induced Duox2 and DuoxA2 expression. A , signaling pathways involved in the regulation of Duox2 expression in BxPC-3 cells by IFN-γ. BxPC-3 cells were

    Techniques Used: Expressing

    p38-mediated Stat1 Ser727 phosphorylation is necessary for IFN-γ-induced Duox2 and DuoxA2 expression. A , IFN-γ activates p38 and ERK as well as Duox in BxPC-3 cells. Time course demonstrating the effect of IFN-γ treatment on ERK
    Figure Legend Snippet: p38-mediated Stat1 Ser727 phosphorylation is necessary for IFN-γ-induced Duox2 and DuoxA2 expression. A , IFN-γ activates p38 and ERK as well as Duox in BxPC-3 cells. Time course demonstrating the effect of IFN-γ treatment on ERK

    Techniques Used: Expressing

    Chromatin immunoprecipitation assay detects Stat1 binding to the Duox2 promoter. Starved BxPC-3 cells were treated with solvent or 25 ng/ml of IFN-γ for 1 or 24 h; cells were then harvested and used for the ChIP assay. Input lanes verifying equal
    Figure Legend Snippet: Chromatin immunoprecipitation assay detects Stat1 binding to the Duox2 promoter. Starved BxPC-3 cells were treated with solvent or 25 ng/ml of IFN-γ for 1 or 24 h; cells were then harvested and used for the ChIP assay. Input lanes verifying equal

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay

    17) Product Images from "High throughput, efficacious gene editing genome surveillance in Chinese hamster ovary cells"

    Article Title: High throughput, efficacious gene editing genome surveillance in Chinese hamster ovary cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0218653

    Comparison of ZFN mRNA vs. CRISPR RNP at the glutamine synthetase locus. Five gRNAs were designed to target GS exon 5. The active site is designated in light red, while the gRNA binding sites are labeled CR1-5 (top left panel) . ZFN binding sites from Fan et al. are indicated as the grey ZFN1 and ZFN2 boxes. Doench scores demonstrating the predicted on-target (cleavage) efficiency as well as the predicted off-target (cleavage of non-target sites) are predicted in silico within the table ( left hand table ). Cells were then transfected with CRISPR RNPs 1 through 5, and gDNA was PCR amplified subjected to a T7 endonuclease assay, with or without addition of nuclease (shown by a + or—; top right hand panel ). Yellow asterisks indicate cleavage products, and successful editing. The most efficacious gRNA, CR3, is boxed in red. CHO cells were then transfected with the indicated amounts of Zinc Finger mRNA or Cas9 CR-3 RNP. Cells were assessed daily for viability (bottom middle panel, D5 6 ZFN 3-10ug P
    Figure Legend Snippet: Comparison of ZFN mRNA vs. CRISPR RNP at the glutamine synthetase locus. Five gRNAs were designed to target GS exon 5. The active site is designated in light red, while the gRNA binding sites are labeled CR1-5 (top left panel) . ZFN binding sites from Fan et al. are indicated as the grey ZFN1 and ZFN2 boxes. Doench scores demonstrating the predicted on-target (cleavage) efficiency as well as the predicted off-target (cleavage of non-target sites) are predicted in silico within the table ( left hand table ). Cells were then transfected with CRISPR RNPs 1 through 5, and gDNA was PCR amplified subjected to a T7 endonuclease assay, with or without addition of nuclease (shown by a + or—; top right hand panel ). Yellow asterisks indicate cleavage products, and successful editing. The most efficacious gRNA, CR3, is boxed in red. CHO cells were then transfected with the indicated amounts of Zinc Finger mRNA or Cas9 CR-3 RNP. Cells were assessed daily for viability (bottom middle panel, D5 6 ZFN 3-10ug P

    Techniques Used: CRISPR, Binding Assay, Labeling, In Silico, Transfection, Polymerase Chain Reaction, Amplification

    18) Product Images from "Rapid Phenotypic Characterization Method for Herpes Simplex Virus and Varicella-Zoster Virus Thymidine Kinases To Screen for Acyclovir-Resistant Viral Infection"

    Article Title: Rapid Phenotypic Characterization Method for Herpes Simplex Virus and Varicella-Zoster Virus Thymidine Kinases To Screen for Acyclovir-Resistant Viral Infection

    Journal: Journal of Clinical Microbiology

    doi:

    Sensitivity of PCR to HSV-1 TK (A), HSV-2 TK (B), and VZV TK (C) genes. The sample DNA, calculated to correspond to 10, 1, 0.1, 0.01, and 0.001 virus-infected cell, was added to 100 μl of PCR mixture, and PCR was carried out under the conditions described in Materials and Methods. After purification of PCR products with a QIAquick PCR Purification Kit, 1 μl of the total 30-μl DNA suspension was analyzed on agarose gels. The amount of VZV TK gene in 1 μl of purified PCR products, amplified from 0.1 infected cell, was measured to be 40 ng by absorbance at 260 nm (panel C, lane 3).
    Figure Legend Snippet: Sensitivity of PCR to HSV-1 TK (A), HSV-2 TK (B), and VZV TK (C) genes. The sample DNA, calculated to correspond to 10, 1, 0.1, 0.01, and 0.001 virus-infected cell, was added to 100 μl of PCR mixture, and PCR was carried out under the conditions described in Materials and Methods. After purification of PCR products with a QIAquick PCR Purification Kit, 1 μl of the total 30-μl DNA suspension was analyzed on agarose gels. The amount of VZV TK gene in 1 μl of purified PCR products, amplified from 0.1 infected cell, was measured to be 40 ng by absorbance at 260 nm (panel C, lane 3).

    Techniques Used: Polymerase Chain Reaction, Infection, Purification, Amplification

    19) Product Images from "Use of a restriction enzyme-digested PCR product as substrate for helicase assays"

    Article Title: Use of a restriction enzyme-digested PCR product as substrate for helicase assays

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni009

    Substrates for helicase assays generated by PCR. A PCR product and its restriction fragment derivatives are shown. PCR reactions were performed as described in Materials and Methods with 32 P-labeled primer, and products were purified using the QIAquick PCR purification kit (Qiagen) (lane 1) and digest with PstI (lane 2), EcoRI (lane 3) and SmaI (lane 4) restriction enzymes.
    Figure Legend Snippet: Substrates for helicase assays generated by PCR. A PCR product and its restriction fragment derivatives are shown. PCR reactions were performed as described in Materials and Methods with 32 P-labeled primer, and products were purified using the QIAquick PCR purification kit (Qiagen) (lane 1) and digest with PstI (lane 2), EcoRI (lane 3) and SmaI (lane 4) restriction enzymes.

    Techniques Used: Generated, Polymerase Chain Reaction, Labeling, Purification

    20) Product Images from "Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier"

    Article Title: Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007486

    Impact of Xrcc4 deletion on Tus/ Ter -induced and I-SceI-induced HR. A , Schematic of 6x Ter -HR reporter and HR repair products of Tus- Ter -induced fork stalling. Green box: wt GFP . Grey boxes: mutant GFP . Open ovals A and B: 5’ and 3’ artificial RFP exons. 5’Tr- GFP : 5’-truncated GFP . Orange triangle: 6x Ter element array. Navy blue line: I-SceI endonuclease cut site. STGC, LTGC: short tract and long tract gene conversion HR repair outcomes. LTGC generates wt RFP through RNA splicing (red filled ovals). B , Xrcc4 gene structure in Xrcc4 fl/fl ES cells. Xrcc4 Δ / Δ allele lacks exon 3. Black triangles: loxP sites. Grey boxes: Xrcc4 Exons 2–4. Location and direction of Exon3 genotyping primers a, a’, and b as indicated by arrows. Gel: PCR products for Xrcc4 fl/fl ES clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13. C , RT qPCR analysis of Xrcc4 expression in Xrcc4 fl/fl or Xrcc4 Δ / Δ clones. Xrcc4 expression normalized to GAPDH and displayed as fold difference from Xrcc4 fl/fl clone 8 of the same experiment (x = -2 ΔΔCt , with ΔΔCt = [Ct Xrcc4 -Ct Gapdh ]-[Ct Xrcc4 -Ct GAPDH ]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV Xrcc4 2 + SDEV GAPDH 2 ]). Xrcc4 abundance by Western blot in Xrcc4 fl/fl clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13 cell protein extracts. D , Representative primary FACS data for two Xrcc4 fl/fl and two Xrcc4 Δ/Δ 6x Ter -HR reporter clones, as indicated, transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. FACS plots produced from pooled data of duplicate samples from three independent experiments. Numbers represent percentages. E , Frequencies of Tus/ Ter -induced and I-SceI-induced repair in five independently derived Xrcc4 fl/fl (orange triangles, red squares) or Xrcc4 Δ/Δ (blue diamonds, navy blue circles) 6x Ter . Error bars: standard error of the mean (s.e.m.). One-way ANOVA (Analysis of Variance) test comparing trend in HR between five Xrcc4 fl/fl and five Xrcc4 Δ/Δ clones: Tus-induced HR, total HR, p = 0.0017; STGC, p = 0.0015; LTGC, p = 0.7142; LTGC/(Total HR), p = 0.2636. I-SceI-induced HR, total HR, p
    Figure Legend Snippet: Impact of Xrcc4 deletion on Tus/ Ter -induced and I-SceI-induced HR. A , Schematic of 6x Ter -HR reporter and HR repair products of Tus- Ter -induced fork stalling. Green box: wt GFP . Grey boxes: mutant GFP . Open ovals A and B: 5’ and 3’ artificial RFP exons. 5’Tr- GFP : 5’-truncated GFP . Orange triangle: 6x Ter element array. Navy blue line: I-SceI endonuclease cut site. STGC, LTGC: short tract and long tract gene conversion HR repair outcomes. LTGC generates wt RFP through RNA splicing (red filled ovals). B , Xrcc4 gene structure in Xrcc4 fl/fl ES cells. Xrcc4 Δ / Δ allele lacks exon 3. Black triangles: loxP sites. Grey boxes: Xrcc4 Exons 2–4. Location and direction of Exon3 genotyping primers a, a’, and b as indicated by arrows. Gel: PCR products for Xrcc4 fl/fl ES clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13. C , RT qPCR analysis of Xrcc4 expression in Xrcc4 fl/fl or Xrcc4 Δ / Δ clones. Xrcc4 expression normalized to GAPDH and displayed as fold difference from Xrcc4 fl/fl clone 8 of the same experiment (x = -2 ΔΔCt , with ΔΔCt = [Ct Xrcc4 -Ct Gapdh ]-[Ct Xrcc4 -Ct GAPDH ]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV Xrcc4 2 + SDEV GAPDH 2 ]). Xrcc4 abundance by Western blot in Xrcc4 fl/fl clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13 cell protein extracts. D , Representative primary FACS data for two Xrcc4 fl/fl and two Xrcc4 Δ/Δ 6x Ter -HR reporter clones, as indicated, transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. FACS plots produced from pooled data of duplicate samples from three independent experiments. Numbers represent percentages. E , Frequencies of Tus/ Ter -induced and I-SceI-induced repair in five independently derived Xrcc4 fl/fl (orange triangles, red squares) or Xrcc4 Δ/Δ (blue diamonds, navy blue circles) 6x Ter . Error bars: standard error of the mean (s.e.m.). One-way ANOVA (Analysis of Variance) test comparing trend in HR between five Xrcc4 fl/fl and five Xrcc4 Δ/Δ clones: Tus-induced HR, total HR, p = 0.0017; STGC, p = 0.0015; LTGC, p = 0.7142; LTGC/(Total HR), p = 0.2636. I-SceI-induced HR, total HR, p

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Clone Assay, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot, FACS, Transfection, Produced, Derivative Assay

    21) Product Images from "Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits"

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58586-3

    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Figure Legend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

    22) Product Images from "The MYC 3′ Wnt-Responsive Element Drives Oncogenic MYC Expression in Human Colorectal Cancer Cells"

    Article Title: The MYC 3′ Wnt-Responsive Element Drives Oncogenic MYC Expression in Human Colorectal Cancer Cells

    Journal: Cancers

    doi: 10.3390/cancers8050052

    Deleting TBE1 reduces TCF7L2/β-catenin binding to the MYC 3′ WRE and MYC expression. ( A ) Diagram of the MYC genomic locus with the positions of PCR amplicons indicated by grey rectangles and labeled 1-5; ( B ) qPCR analysis of DNA fragments precipitated with anti-TCF7L2 antibodies in ChIP assays conducted in control and 3′ WRE-Mut cells. Shown are signals obtained from PCR amplicons listed on the x-axis. The data is normalized to levels detected with amplicon 1; ( C ) as in ( B ) except anti-β-catenin antibodies were used in the ChIP assays; ( D ) qRT-PCR analysis of MYC transcript levels detected in control and 3′ WRE-Mut cells. The data is normalized to GAPDH levels; ( E ) Western blot analysis of MYC protein levels in control and 3′ WRE-Mut cells. The blots were re-probed with anti-α-tubulin antibodies to control for loading. Shown below are relative levels of MYC, which were quantified using densitometry. Error bars are ±SEM (*** p
    Figure Legend Snippet: Deleting TBE1 reduces TCF7L2/β-catenin binding to the MYC 3′ WRE and MYC expression. ( A ) Diagram of the MYC genomic locus with the positions of PCR amplicons indicated by grey rectangles and labeled 1-5; ( B ) qPCR analysis of DNA fragments precipitated with anti-TCF7L2 antibodies in ChIP assays conducted in control and 3′ WRE-Mut cells. Shown are signals obtained from PCR amplicons listed on the x-axis. The data is normalized to levels detected with amplicon 1; ( C ) as in ( B ) except anti-β-catenin antibodies were used in the ChIP assays; ( D ) qRT-PCR analysis of MYC transcript levels detected in control and 3′ WRE-Mut cells. The data is normalized to GAPDH levels; ( E ) Western blot analysis of MYC protein levels in control and 3′ WRE-Mut cells. The blots were re-probed with anti-α-tubulin antibodies to control for loading. Shown below are relative levels of MYC, which were quantified using densitometry. Error bars are ±SEM (*** p

    Techniques Used: Binding Assay, Expressing, Polymerase Chain Reaction, Labeling, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR, Western Blot

    23) Product Images from "Generation of shape complexity through tissue conflict resolution"

    Article Title: Generation of shape complexity through tissue conflict resolution

    Journal: eLife

    doi: 10.7554/eLife.20156

    Expression patterns of AmPIN1 genes. ( A-L ) Expression patterns of AmPIN1 genes at 8 DAI ( A-C ), 10 DAI ( D-F ), 12 DAI ( G-I ) and 14 DAI ( J-L ), determined by RNA in situ hybridisation of alternative sections hybridised with AmPIN1a (left panels, A , D , G , J ), AmPIN1b (middle panels, B , E , H , K ) and AmPIN1c (right panels, C , F , I , L ) probes. The AmPIN1 genes show similar expression patterns in the vascular tissue of petal primordia ( A-F ), with some localised differences particularly at the tips of the petal primordia ( D-F and close-ups of red boxes in respective D-F images), in particular for AmPIN1c which shows stronger epidermal expression at the petal tips. At 12 DAI, the AmPIN1 genes continue to be expressed in the vascular tissue ( G-I ) but are additionally detected at the epidermis of the palate and lip regions, particularly strongly for AmPIN1c (white arrows in close-ups of blue boxes in respective G-I images). At 13 DAI, the epidermal Am PIN1 expression starts to decrease ( J-L ) in comparison with the vascular tissue signal which continues to be similar to previous stages (close-ups of green squares in respective J-L images). The upper and lower limits of the palate and lip domains are marked by pink dashed lines. The diagram in the upper right corner of every left panel represents the orientation of the section (white line) relative to the five petals (red-dorsal, orange -lateral and yellow -ventral). V: ventral petal; D: dorsal petal; st: stamen; ca: carpel. Scale bars, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.20156.028
    Figure Legend Snippet: Expression patterns of AmPIN1 genes. ( A-L ) Expression patterns of AmPIN1 genes at 8 DAI ( A-C ), 10 DAI ( D-F ), 12 DAI ( G-I ) and 14 DAI ( J-L ), determined by RNA in situ hybridisation of alternative sections hybridised with AmPIN1a (left panels, A , D , G , J ), AmPIN1b (middle panels, B , E , H , K ) and AmPIN1c (right panels, C , F , I , L ) probes. The AmPIN1 genes show similar expression patterns in the vascular tissue of petal primordia ( A-F ), with some localised differences particularly at the tips of the petal primordia ( D-F and close-ups of red boxes in respective D-F images), in particular for AmPIN1c which shows stronger epidermal expression at the petal tips. At 12 DAI, the AmPIN1 genes continue to be expressed in the vascular tissue ( G-I ) but are additionally detected at the epidermis of the palate and lip regions, particularly strongly for AmPIN1c (white arrows in close-ups of blue boxes in respective G-I images). At 13 DAI, the epidermal Am PIN1 expression starts to decrease ( J-L ) in comparison with the vascular tissue signal which continues to be similar to previous stages (close-ups of green squares in respective J-L images). The upper and lower limits of the palate and lip domains are marked by pink dashed lines. The diagram in the upper right corner of every left panel represents the orientation of the section (white line) relative to the five petals (red-dorsal, orange -lateral and yellow -ventral). V: ventral petal; D: dorsal petal; st: stamen; ca: carpel. Scale bars, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.20156.028

    Techniques Used: Expressing, In Situ, Hybridization

    24) Product Images from "RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples"

    Article Title: RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh116

    Hybridization of a set of standard liver cDNA mixtures containing increasing amounts of bacterial LysA antisense cDNA. ( A ) LysA abundance varying from approximately 9 to 9100 copies LysA cDNA per cell equivalent are shown in this figure (left vertical
    Figure Legend Snippet: Hybridization of a set of standard liver cDNA mixtures containing increasing amounts of bacterial LysA antisense cDNA. ( A ) LysA abundance varying from approximately 9 to 9100 copies LysA cDNA per cell equivalent are shown in this figure (left vertical

    Techniques Used: Hybridization

    25) Product Images from "Assessment of efficacy of prenatal genetic diagnosis for fragile X syndrome using nested PCR"

    Article Title: Assessment of efficacy of prenatal genetic diagnosis for fragile X syndrome using nested PCR

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2018.6060

    Detection from spiked blood spots. (A) Nested PCR screens expansion mutation of FMR1 gene. (B) Nested PCR screens no positive call from the blood spot sample spiked in healthy individuals. PCR, polymerase chain reaction; FMR1, fragile X mental retardation 1.
    Figure Legend Snippet: Detection from spiked blood spots. (A) Nested PCR screens expansion mutation of FMR1 gene. (B) Nested PCR screens no positive call from the blood spot sample spiked in healthy individuals. PCR, polymerase chain reaction; FMR1, fragile X mental retardation 1.

    Techniques Used: Nested PCR, Mutagenesis, Polymerase Chain Reaction

    Comparison of nested PCR and PCR in diagnosis of FXS. (A) Patients with FXS shows lowed FMR1 gene expression compared to healthy individuals. (B and C) The efficacy of traditional PCR (B) and nested PCR (C) for diagnose patients with FXS. (D) Nested PCR presents more sensitive than PCR in diagnosing of FXS. PCR, polymerase chain reaction; FXS, fragile X syndrome; FMR1, fragile X mental retardation 1. **P
    Figure Legend Snippet: Comparison of nested PCR and PCR in diagnosis of FXS. (A) Patients with FXS shows lowed FMR1 gene expression compared to healthy individuals. (B and C) The efficacy of traditional PCR (B) and nested PCR (C) for diagnose patients with FXS. (D) Nested PCR presents more sensitive than PCR in diagnosing of FXS. PCR, polymerase chain reaction; FXS, fragile X syndrome; FMR1, fragile X mental retardation 1. **P

    Techniques Used: Nested PCR, Polymerase Chain Reaction, Expressing

    26) Product Images from "Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9"

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36736-y

    Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.
    Figure Legend Snippet: Efficient targeting of Bmal1 and Per2 genes in U2OS cells by the all-in-one CRISPR-Cas9 vector. ( A ) Four exons were targeted in each gene based on their score in a predictive software tool. The PAM sequences are indicated in red. Note that target sites for Bmal1 exon 6, 7 and 9 are close to splicing sites indicated by dashes within the sequence. ( B ) mCherry-expressing cells (Pos) were selected by FACS and subjected to T7E1 assay to assess the efficiency of indels. Control cells (Neg) were from the same FACS sorting. Cells with low mCherry signal (middle) were discarded. All of the eight samples showed similarly efficient indels, proportional to transfection efficiency. Two representative samples are shown. ( C ) Targeting of the clock genes by the all-in-one vectors produces diverse indels including frame-shifting mutations. PCR amplicons from the genomic targets of Bmal1 exon 7 and Per2 exon 15 were sequenced (see Supplementary Fig. 1 for Per2 exon 15). 15–30% of clones were wt. Deletions and insertions are indicated by lines and green characters, respectively.

    Techniques Used: CRISPR, Plasmid Preparation, Software, Sequencing, Expressing, FACS, Transfection, Polymerase Chain Reaction, Clone Assay

    Generation of an all-in-one Cas9-mCherry-sgRNA vector using the Gateway system. ( A ) A Gateway entry plasmid was generated to clone a specific sgRNA into a PCR amplicon flanked by attL1 and attL2. PCR from the Entry vector using two sets of primers with specific 20 nt gRNA sequence (reaction 1 and 2) produces two fragments that partially overlap and can be combined by an overlap extension PCR (reaction 3). Multiplexing is possible by using multiple sets of Scaff-fwd and U6-Rev primers. ( B ) PCR products of two individual fragments and a full-length L1-U6-sgRNA-L2 are shown. ( C ) The final PCR amplicons can be combined with the adenoviral shuttle vector with R1-ccdB-R2 Destination sequence or used separately. pShuttle-Cas9-DEST was generated by cloning Cas9-mCherry and R1-R2 into pShuttle. ( D ) Gateway LR cloning produces 100% positive clones. Since the background (before recombination) clones cannot grow in ccdB-incompatible bacterial cell lines such as DH5a, all the transformed colonies with the LR reaction mixture contain the recombinant without ccdB, but with sgRNA. Two independent experiments are shown.
    Figure Legend Snippet: Generation of an all-in-one Cas9-mCherry-sgRNA vector using the Gateway system. ( A ) A Gateway entry plasmid was generated to clone a specific sgRNA into a PCR amplicon flanked by attL1 and attL2. PCR from the Entry vector using two sets of primers with specific 20 nt gRNA sequence (reaction 1 and 2) produces two fragments that partially overlap and can be combined by an overlap extension PCR (reaction 3). Multiplexing is possible by using multiple sets of Scaff-fwd and U6-Rev primers. ( B ) PCR products of two individual fragments and a full-length L1-U6-sgRNA-L2 are shown. ( C ) The final PCR amplicons can be combined with the adenoviral shuttle vector with R1-ccdB-R2 Destination sequence or used separately. pShuttle-Cas9-DEST was generated by cloning Cas9-mCherry and R1-R2 into pShuttle. ( D ) Gateway LR cloning produces 100% positive clones. Since the background (before recombination) clones cannot grow in ccdB-incompatible bacterial cell lines such as DH5a, all the transformed colonies with the LR reaction mixture contain the recombinant without ccdB, but with sgRNA. Two independent experiments are shown.

    Techniques Used: Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Sequencing, Multiplexing, Clone Assay, Transformation Assay, Recombinant

    27) Product Images from "Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication"

    Article Title: Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

    Journal: bioRxiv

    doi: 10.1101/789578

    Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on λ DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on λ DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647 signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on λ DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on λ DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on λ DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on λ DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on λ DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on λ DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.
    Figure Legend Snippet: Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on λ DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on λ DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647 signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on λ DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on λ DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on λ DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on λ DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on λ DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on λ DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.

    Techniques Used: Staining, Fluorescence, Migration, Imaging, Concentration Assay

    Histone dynamics during DNA licensing in HSS. (A) Schematic of the experimental set-up for real-time single-molecule imaging of nucleosome dynamics during replication in Xenopus leavis egg extracts. λ DNA containing fluorescent nucleosomes (one of the four histones labelled fluorescently) is stretched under flow and tethered at both ends to the functionalized glass surface of a microfluidic flow cell. The immobilized DNA is licensed in high-speed supernatant (HSS). Bidirectional replication is initiated upon introduction of nucleoplasmic extract (NPE) supplemented with a fluorescent fusion protein Fen1-KikGR, which decorates replication bubbles and allows progression of replication forks to be tracked in real time. Cy5- or Alexa647-labelled histones within immobilized nucleosomal templates are imaged with a 640-nm laser at each stage. Replication fork progression is visualized in NPE using a 488-nm laser. (B and C) Kymograms and corresponding intensity profiles for fluorescent λ nucleosomes during incubation in HSS. Nucleosomes labelled at H2A-K119C with Cy5 and H2B-T112C with AlexaFluor647 (B) show faster loss of fluorescence than nucleosomes labelled at H3-K36C with Cy5, H3-T80C with AlexaFluor647 and H4-E63C with AlexaFluor647 (C). (D) Plot showing the mean loss of fluorescent signal for λ nucleosomes (H2A-K119 Cy5 , H2B-T112C A647 , H3-K36C Cy5 , H3-T80C A647 and H4-E63C A647 ) during incubation in HSS. Over 100 molecules were analyzed for each histone template. Individual fluorescence decay traces were normalized to background (‘0’) and maximum value of fluorescence (‘1’). A mean fluorescence value and standard deviation were calculated and plotted for each time point. The mean value traces were then fitted to a single exponential function. (E) Summary of the fluorescence decay rate constants ( K ) and half-lives ( t 0.5) extracted from the single exponential fit to the data presented in panel C. See Table S2 for detailed fitting parameters.
    Figure Legend Snippet: Histone dynamics during DNA licensing in HSS. (A) Schematic of the experimental set-up for real-time single-molecule imaging of nucleosome dynamics during replication in Xenopus leavis egg extracts. λ DNA containing fluorescent nucleosomes (one of the four histones labelled fluorescently) is stretched under flow and tethered at both ends to the functionalized glass surface of a microfluidic flow cell. The immobilized DNA is licensed in high-speed supernatant (HSS). Bidirectional replication is initiated upon introduction of nucleoplasmic extract (NPE) supplemented with a fluorescent fusion protein Fen1-KikGR, which decorates replication bubbles and allows progression of replication forks to be tracked in real time. Cy5- or Alexa647-labelled histones within immobilized nucleosomal templates are imaged with a 640-nm laser at each stage. Replication fork progression is visualized in NPE using a 488-nm laser. (B and C) Kymograms and corresponding intensity profiles for fluorescent λ nucleosomes during incubation in HSS. Nucleosomes labelled at H2A-K119C with Cy5 and H2B-T112C with AlexaFluor647 (B) show faster loss of fluorescence than nucleosomes labelled at H3-K36C with Cy5, H3-T80C with AlexaFluor647 and H4-E63C with AlexaFluor647 (C). (D) Plot showing the mean loss of fluorescent signal for λ nucleosomes (H2A-K119 Cy5 , H2B-T112C A647 , H3-K36C Cy5 , H3-T80C A647 and H4-E63C A647 ) during incubation in HSS. Over 100 molecules were analyzed for each histone template. Individual fluorescence decay traces were normalized to background (‘0’) and maximum value of fluorescence (‘1’). A mean fluorescence value and standard deviation were calculated and plotted for each time point. The mean value traces were then fitted to a single exponential function. (E) Summary of the fluorescence decay rate constants ( K ) and half-lives ( t 0.5) extracted from the single exponential fit to the data presented in panel C. See Table S2 for detailed fitting parameters.

    Techniques Used: Imaging, Incubation, Fluorescence, Standard Deviation

    Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the Xenopus nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked λ DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal λ templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked λ DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (
    Figure Legend Snippet: Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the Xenopus nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked λ DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal λ templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked λ DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (

    Techniques Used: SDS Page, Staining, Fluorescence, Electrophoretic Mobility Shift Assay

    28) Product Images from "VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism"

    Article Title: VSIG4 inhibits proinflammatory macrophage activation by reprogramming mitochondrial pyruvate metabolism

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01327-4

    Vsig4 gene transcription is repressed Dnmt3a-mediated DNA methylation. The C57BL/6 WT mice were infected with MHV-3 (100 PFU/mouse), a the expression of VSIG4 on PEMs at 0 h and 12 h PI was detected by flow cytometry. b VSIG4 protein level in liver tissues was analyzed by western blot. The BMDMs were treated with proinflammatory factors for 12 h. c Vsig4 gene transcription was detected by qRT-PCR. d VSIG4 protein expression was evaluated by western blot. e The BMDMs were treated with Dnmts inhibitor-AZAdC (10 μM) for 72 h in advance, cells were then further added with proinflammatory mediators for 12 h, the expression of Dnmt3a and VSIG4 was assessed by western blotting. f Luciferase activity of the lysates from RAW264.7 cells transfected with unmethylated or M.SssI methylated pGL3-basic vector and the -840/+1 Vsig4 promoter constructs. Error bar, s.e.m. * p
    Figure Legend Snippet: Vsig4 gene transcription is repressed Dnmt3a-mediated DNA methylation. The C57BL/6 WT mice were infected with MHV-3 (100 PFU/mouse), a the expression of VSIG4 on PEMs at 0 h and 12 h PI was detected by flow cytometry. b VSIG4 protein level in liver tissues was analyzed by western blot. The BMDMs were treated with proinflammatory factors for 12 h. c Vsig4 gene transcription was detected by qRT-PCR. d VSIG4 protein expression was evaluated by western blot. e The BMDMs were treated with Dnmts inhibitor-AZAdC (10 μM) for 72 h in advance, cells were then further added with proinflammatory mediators for 12 h, the expression of Dnmt3a and VSIG4 was assessed by western blotting. f Luciferase activity of the lysates from RAW264.7 cells transfected with unmethylated or M.SssI methylated pGL3-basic vector and the -840/+1 Vsig4 promoter constructs. Error bar, s.e.m. * p

    Techniques Used: DNA Methylation Assay, Mouse Assay, Infection, Expressing, Flow Cytometry, Cytometry, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Methylation, Plasmid Preparation, Construct

    29) Product Images from "Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors"

    Article Title: Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

    Journal: Nature Communications

    doi: 10.1038/ncomms6750

    Genomic analysis of a deleted region in the ferret CMAH gene responsible for the absence of Neu5Gc in both ferrets and humans. ( a ) Synteny in the CMAH region of mouse, human and cat genomes. ( b ) Representation of the CMAH region of the ferret genome characterized in this study. Black bars represent region spanned by BAC clones 446P7 and 182P23. Blue bars indicate location of probes used in screening BAC library. Arrows indicate position of primers used to amplify the PCR product spanning the CMAH -deletion region. ( c ) Comparison of the CMAH region in cat and ferret genomes. Seven-kb PCR product containing the CMAH -deleted region was amplified using primers CMAH_FOR and CMAH_REV, respectively. (*) indicates the start of the CMAH gene, (**) indicates individual exon(s) on the CMAH gene and (***) indicates the end of the CMAH gene. Images below show CMAH exon PCR products from exon 3, exon 5, exon 8, exon 11 and exon 12, from cat (C), dog (D), human (H) and ferret (F) genomic DNA.
    Figure Legend Snippet: Genomic analysis of a deleted region in the ferret CMAH gene responsible for the absence of Neu5Gc in both ferrets and humans. ( a ) Synteny in the CMAH region of mouse, human and cat genomes. ( b ) Representation of the CMAH region of the ferret genome characterized in this study. Black bars represent region spanned by BAC clones 446P7 and 182P23. Blue bars indicate location of probes used in screening BAC library. Arrows indicate position of primers used to amplify the PCR product spanning the CMAH -deletion region. ( c ) Comparison of the CMAH region in cat and ferret genomes. Seven-kb PCR product containing the CMAH -deleted region was amplified using primers CMAH_FOR and CMAH_REV, respectively. (*) indicates the start of the CMAH gene, (**) indicates individual exon(s) on the CMAH gene and (***) indicates the end of the CMAH gene. Images below show CMAH exon PCR products from exon 3, exon 5, exon 8, exon 11 and exon 12, from cat (C), dog (D), human (H) and ferret (F) genomic DNA.

    Techniques Used: BAC Assay, Clone Assay, Polymerase Chain Reaction, Amplification

    30) Product Images from "The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer"

    Article Title: The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky331

    HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.
    Figure Legend Snippet: HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Ligation, Purification, Amplification, Polymerase Chain Reaction, Sequencing, Derivative Assay

    31) Product Images from "Enhancing mammary differentiation by overcoming lineage-specific epigenetic modification and signature gene expression of fibroblast-derived iPSCs"

    Article Title: Enhancing mammary differentiation by overcoming lineage-specific epigenetic modification and signature gene expression of fibroblast-derived iPSCs

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.499

    Analysis of gene expression and epigenetic modification of iPSCs. ( a ) Validation of the differential expression of selected genes by qRT-PCR. Relative levels in D-ME-iPSCs and ME cells were compared with the level in D-TF-iPSCs, which was set at 1. ( b – d ) Epigenetic modification in the promoter of Elf5, Cldn1, and vimentin in ME cells, D-ME-iPSCs, D-TF-iPSCs, and TFs revealed by ChIP analysis using the antibodies indicated. The P -values are as indicated. ( e ) qPCR analysis of the methylation status of Cldn1 , 3 , 4 , and 7 using specific primers for methylated (M) and unmethylated (U) DNA in ME cells, D-ME-iPSCs, and D-TF-iPSCs. ( f ) Bisulfite sequencing of Cldn3 . * P
    Figure Legend Snippet: Analysis of gene expression and epigenetic modification of iPSCs. ( a ) Validation of the differential expression of selected genes by qRT-PCR. Relative levels in D-ME-iPSCs and ME cells were compared with the level in D-TF-iPSCs, which was set at 1. ( b – d ) Epigenetic modification in the promoter of Elf5, Cldn1, and vimentin in ME cells, D-ME-iPSCs, D-TF-iPSCs, and TFs revealed by ChIP analysis using the antibodies indicated. The P -values are as indicated. ( e ) qPCR analysis of the methylation status of Cldn1 , 3 , 4 , and 7 using specific primers for methylated (M) and unmethylated (U) DNA in ME cells, D-ME-iPSCs, and D-TF-iPSCs. ( f ) Bisulfite sequencing of Cldn3 . * P

    Techniques Used: Expressing, Modification, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylation, Methylation Sequencing

    32) Product Images from "Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics"

    Article Title: Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics

    Journal: Virology Journal

    doi: 10.1186/s12985-016-0504-8

    Comparison of selected treatments on the visualization of RPA-generated amplicons. a Results using primer pair TYL828F/TYL834R from crude extracts prepared in 0.5 N NaOH from Tomato yellow leaf curl virus (TYLCV)-infected tomato leaves and cleaned by heating to 65 °C for 15 min. Lane M: 50 bp ladder MW standard, size is indicated in kilobases (kb); Lane 1 fresh tissue; Lane 2 tissue kept frozen at −20 °C for 5 months; Lane 3 tissue kept frozen at −80 °C for 3 months; Lane 4 desiccated tissue maintained at room temperature for 15 years; Lane 5 non-inoculated tissue kept frozen at −80 °C for 3 months. b Detection of TYLCV from crude extracts prepared in 0.5 N NaOH and cleaned as follows: Lane 1 untreated; Lane 2 QIAquick PCR purification column; Lane 3 heated at 65 °C for 10 min; Lane 4 heated at 95 °C for 10 min; Lane 5 amplicon loading buffer contained 5 % SDS; Lane 6 amplicon loading buffer contained 10 % SDS; Lane 7 amplicon loading buffer contained 5 % formamide; Lane 8 amplicon loading buffer contained 15 % formamide. Ten μl of amplified product were loaded into each lane of the 1.5 % agarose gels and stained with ethidium bromide
    Figure Legend Snippet: Comparison of selected treatments on the visualization of RPA-generated amplicons. a Results using primer pair TYL828F/TYL834R from crude extracts prepared in 0.5 N NaOH from Tomato yellow leaf curl virus (TYLCV)-infected tomato leaves and cleaned by heating to 65 °C for 15 min. Lane M: 50 bp ladder MW standard, size is indicated in kilobases (kb); Lane 1 fresh tissue; Lane 2 tissue kept frozen at −20 °C for 5 months; Lane 3 tissue kept frozen at −80 °C for 3 months; Lane 4 desiccated tissue maintained at room temperature for 15 years; Lane 5 non-inoculated tissue kept frozen at −80 °C for 3 months. b Detection of TYLCV from crude extracts prepared in 0.5 N NaOH and cleaned as follows: Lane 1 untreated; Lane 2 QIAquick PCR purification column; Lane 3 heated at 65 °C for 10 min; Lane 4 heated at 95 °C for 10 min; Lane 5 amplicon loading buffer contained 5 % SDS; Lane 6 amplicon loading buffer contained 10 % SDS; Lane 7 amplicon loading buffer contained 5 % formamide; Lane 8 amplicon loading buffer contained 15 % formamide. Ten μl of amplified product were loaded into each lane of the 1.5 % agarose gels and stained with ethidium bromide

    Techniques Used: Recombinase Polymerase Amplification, Generated, Infection, Polymerase Chain Reaction, Purification, Amplification, Staining

    33) Product Images from "Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics"

    Article Title: Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics

    Journal: Virology Journal

    doi: 10.1186/s12985-016-0504-8

    Comparison of selected treatments on the visualization of RPA-generated amplicons. a Results using primer pair TYL828F/TYL834R from crude extracts prepared in 0.5 N NaOH from Tomato yellow leaf curl virus (TYLCV)-infected tomato leaves and cleaned by heating to 65 °C for 15 min. Lane M: 50 bp ladder MW standard, size is indicated in kilobases (kb); Lane 1 fresh tissue; Lane 2 tissue kept frozen at −20 °C for 5 months; Lane 3 tissue kept frozen at −80 °C for 3 months; Lane 4 desiccated tissue maintained at room temperature for 15 years; Lane 5 non-inoculated tissue kept frozen at −80 °C for 3 months. b Detection of TYLCV from crude extracts prepared in 0.5 N NaOH and cleaned as follows: Lane 1 untreated; Lane 2 QIAquick PCR purification column; Lane 3 heated at 65 °C for 10 min; Lane 4 heated at 95 °C for 10 min; Lane 5 amplicon loading buffer contained 5 % SDS; Lane 6 amplicon loading buffer contained 10 % SDS; Lane 7 amplicon loading buffer contained 5 % formamide; Lane 8 amplicon loading buffer contained 15 % formamide. Ten μl of amplified product were loaded into each lane of the 1.5 % agarose gels and stained with ethidium bromide
    Figure Legend Snippet: Comparison of selected treatments on the visualization of RPA-generated amplicons. a Results using primer pair TYL828F/TYL834R from crude extracts prepared in 0.5 N NaOH from Tomato yellow leaf curl virus (TYLCV)-infected tomato leaves and cleaned by heating to 65 °C for 15 min. Lane M: 50 bp ladder MW standard, size is indicated in kilobases (kb); Lane 1 fresh tissue; Lane 2 tissue kept frozen at −20 °C for 5 months; Lane 3 tissue kept frozen at −80 °C for 3 months; Lane 4 desiccated tissue maintained at room temperature for 15 years; Lane 5 non-inoculated tissue kept frozen at −80 °C for 3 months. b Detection of TYLCV from crude extracts prepared in 0.5 N NaOH and cleaned as follows: Lane 1 untreated; Lane 2 QIAquick PCR purification column; Lane 3 heated at 65 °C for 10 min; Lane 4 heated at 95 °C for 10 min; Lane 5 amplicon loading buffer contained 5 % SDS; Lane 6 amplicon loading buffer contained 10 % SDS; Lane 7 amplicon loading buffer contained 5 % formamide; Lane 8 amplicon loading buffer contained 15 % formamide. Ten μl of amplified product were loaded into each lane of the 1.5 % agarose gels and stained with ethidium bromide

    Techniques Used: Recombinase Polymerase Amplification, Generated, Infection, Polymerase Chain Reaction, Purification, Amplification, Staining

    34) Product Images from "An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition"

    Article Title: An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166438

    Reduced H3K27me3 binding is detected by ChIP-qPCR. (A) ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in the presence of the inhibitor. (B) ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in cells treated with the inhibitor. ( C ) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for MYT1 and CCND2 . ( D ) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for MYT1 and CCND2 . All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The ACTB promoter served as a negative control for all experiments.
    Figure Legend Snippet: Reduced H3K27me3 binding is detected by ChIP-qPCR. (A) ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in the presence of the inhibitor. (B) ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in cells treated with the inhibitor. ( C ) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for MYT1 and CCND2 . ( D ) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for MYT1 and CCND2 . All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The ACTB promoter served as a negative control for all experiments.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Generated, Polymerase Chain Reaction, Amplification, Negative Control

    35) Product Images from "DNA methylation of E-cadherin is a priming mechanism for prostate development"

    Article Title: DNA methylation of E-cadherin is a priming mechanism for prostate development

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2014.01.020

    DNA methylation is required for appropriate E-cadherin mRNA and protein expression in basal epithelium of UGS explant cultures. (A) MeDIP-QPCR was used to quantify E-cadherin ( Cdh1 ) methylation in epithelium from 14 dpc UGSs cultured for 4 days in media
    Figure Legend Snippet: DNA methylation is required for appropriate E-cadherin mRNA and protein expression in basal epithelium of UGS explant cultures. (A) MeDIP-QPCR was used to quantify E-cadherin ( Cdh1 ) methylation in epithelium from 14 dpc UGSs cultured for 4 days in media

    Techniques Used: DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylation, Cell Culture

    36) Product Images from "Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers"

    Article Title: Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers

    Journal: Current protocols in microbiology

    doi: 10.1002/9780471729259.mc01e04s20

    Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.
    Figure Legend Snippet: Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.

    Techniques Used: Ligation, Polymerase Chain Reaction

    Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis , and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.
    Figure Legend Snippet: Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis , and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.

    Techniques Used: Sequencing, Purification, Modification, Polymerase Chain Reaction

    37) Product Images from "Improved Protocols for Illumina Sequencing"

    Article Title: Improved Protocols for Illumina Sequencing

    Journal: Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]

    doi: 10.1002/0471142905.hg1802s62

    Post-sequencing mapped insert-size distribution graphs for Illumina sequencing libraries prepared from genomic DNA with size-selection using; (A) agarose gel electrophoresis, (B) AMPure XP, (C) Caliper Labchip XT and (D) Sage Science Pippin Prep.
    Figure Legend Snippet: Post-sequencing mapped insert-size distribution graphs for Illumina sequencing libraries prepared from genomic DNA with size-selection using; (A) agarose gel electrophoresis, (B) AMPure XP, (C) Caliper Labchip XT and (D) Sage Science Pippin Prep.

    Techniques Used: Sequencing, Selection, Agarose Gel Electrophoresis

    ( A ) Template hybridization, extension, and denaturation on the flowcell surface. Templates are prepared so as to possess tails that are complementary to primers on the flowcell surface. This allows one end of a template strand to hybridize to a flowcell primer. Flowcell primers are extended by Phusion DNA polymerase (Thermo Scientific), resulting in a reverse complementary copy of the original template strand, which is covalently attached to the flowcell surface. The original template strand is then removed by flushing 0.1 M NaOH though the flowcell. ( B ) Cluster amplification. The free end of the tethered reverse complementary copy of the original template strand can anneal to the other type of flowcell primer, forming a bridge. The flowcell primer is extended by Bst polymerase, in an isothermal reaction, which generates a double-stranded product. Formamide is used to denature these strands, which can then anneal to other primers on the flowcell surface, which extend in the next cycle. In this way, repeated cycles of extension and denaturation result in a cluster of strands, all of which are derived from a single template strand.
    Figure Legend Snippet: ( A ) Template hybridization, extension, and denaturation on the flowcell surface. Templates are prepared so as to possess tails that are complementary to primers on the flowcell surface. This allows one end of a template strand to hybridize to a flowcell primer. Flowcell primers are extended by Phusion DNA polymerase (Thermo Scientific), resulting in a reverse complementary copy of the original template strand, which is covalently attached to the flowcell surface. The original template strand is then removed by flushing 0.1 M NaOH though the flowcell. ( B ) Cluster amplification. The free end of the tethered reverse complementary copy of the original template strand can anneal to the other type of flowcell primer, forming a bridge. The flowcell primer is extended by Bst polymerase, in an isothermal reaction, which generates a double-stranded product. Formamide is used to denature these strands, which can then anneal to other primers on the flowcell surface, which extend in the next cycle. In this way, repeated cycles of extension and denaturation result in a cluster of strands, all of which are derived from a single template strand.

    Techniques Used: Hybridization, Amplification, Derivative Assay

    Illumina flowcell. ( A ) Flowcells are hollow glass slides, with 8 separate lanes, through which reagents and template DNA flow. Lanes have been shaded gray for clarity. ( B ) Cross-section of a single lane, showing the direction of reagent flow and polyacrylamide coating on the interior surface of the flowcell.
    Figure Legend Snippet: Illumina flowcell. ( A ) Flowcells are hollow glass slides, with 8 separate lanes, through which reagents and template DNA flow. Lanes have been shaded gray for clarity. ( B ) Cross-section of a single lane, showing the direction of reagent flow and polyacrylamide coating on the interior surface of the flowcell.

    Techniques Used: Flow Cytometry

    38) Product Images from "Northern lights assay: a versatile method for comprehensive detection of DNA damage"

    Article Title: Northern lights assay: a versatile method for comprehensive detection of DNA damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky645

    Cisplatin crosslinks in DNA detected with NLA. ( A ) Analysis of DNA crosslinks in a complex DNA sample. (a) Untreated but Mbo I-digested human genomic DNA (green smear) migrated with the double-stranded Cy5-labeled marker. (b) The same DNA after incubation at 37°C for 16 h showing slight heat-induced damage. (c–e) DNA samples treated with the indicated concentration of cisplatin. Quantification of fractions of duplicate experiments with standard deviations (SD) is shown. A significant increase in DNA migrating behind and in front of undamaged DNA was detected after treatment with all concentrations of cisplatin ( P
    Figure Legend Snippet: Cisplatin crosslinks in DNA detected with NLA. ( A ) Analysis of DNA crosslinks in a complex DNA sample. (a) Untreated but Mbo I-digested human genomic DNA (green smear) migrated with the double-stranded Cy5-labeled marker. (b) The same DNA after incubation at 37°C for 16 h showing slight heat-induced damage. (c–e) DNA samples treated with the indicated concentration of cisplatin. Quantification of fractions of duplicate experiments with standard deviations (SD) is shown. A significant increase in DNA migrating behind and in front of undamaged DNA was detected after treatment with all concentrations of cisplatin ( P

    Techniques Used: Labeling, Marker, Incubation, Concentration Assay

    39) Product Images from "Induction of innate immune memory via microRNA targeting of chromatin remodeling factors"

    Article Title: Induction of innate immune memory via microRNA targeting of chromatin remodeling factors

    Journal: Nature

    doi: 10.1038/s41586-018-0253-5

    Tnf is a direct target of miR-222, but suppression of Tnf does not account for miR-222-mediated transcriptional silencing of late LPS response genes a , Sequence and prediction scores of a miR-222 binding site in the Tnf UTR. b , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Tnf UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (a) (n=6 independent experiments). c , CRISPR-Cas9 targeting strategy to delete predicted binding sites. d , RAW clones were screened for successful deletion of the miR-222 binding site by PCR across the targeted region of the UTR, using genomic DNA from the given clonal line as a template. Screening for Tnf UTR deletion is shown. Experiment was repeated twice with similar results. e , Successful deletion of the miR-222 binding site in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site in the TNF UTR is highlighted in yellow. f , LPS-induced Tnf expression in control and CRISPR-Cas9 targeted RAW cells (n=4 independent experiments). g , Average effect of miR-222 mimic transfection on LPS-induced Tnf mRNA levels in either control MEFs or MEFs which have undergone CRISPR targeting and clonal selection for deletion of the miR-222 binding site. Average of the effects from the 3 clonal lines (n=3 independent experiments). h , Wildtype BMDMs were transfected with a control or miR-222 mimic oligonucleotide. 24 hours later, cells were pre-treated with an isotype control (IgG) or TNF neutralizing (α-TNF) antibody for two hours, and stimulated with 10 ng/ml LPS. Expression of the given genes was measured by qPCR (n=4 biologically independent samples). i , Efficacy of TNF neutralization was confirmed by treating cells with IgG or α-TNF as above, followed by stimulation with 100 ng/ml recombinant mouse TNF (n=3 biologically independent samples). Gene upregulation was not detected (ND) in 2/3 samples treated with α-TNF. For all bar graphs, mean +/− SEM is plotted. ** p
    Figure Legend Snippet: Tnf is a direct target of miR-222, but suppression of Tnf does not account for miR-222-mediated transcriptional silencing of late LPS response genes a , Sequence and prediction scores of a miR-222 binding site in the Tnf UTR. b , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Tnf UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (a) (n=6 independent experiments). c , CRISPR-Cas9 targeting strategy to delete predicted binding sites. d , RAW clones were screened for successful deletion of the miR-222 binding site by PCR across the targeted region of the UTR, using genomic DNA from the given clonal line as a template. Screening for Tnf UTR deletion is shown. Experiment was repeated twice with similar results. e , Successful deletion of the miR-222 binding site in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site in the TNF UTR is highlighted in yellow. f , LPS-induced Tnf expression in control and CRISPR-Cas9 targeted RAW cells (n=4 independent experiments). g , Average effect of miR-222 mimic transfection on LPS-induced Tnf mRNA levels in either control MEFs or MEFs which have undergone CRISPR targeting and clonal selection for deletion of the miR-222 binding site. Average of the effects from the 3 clonal lines (n=3 independent experiments). h , Wildtype BMDMs were transfected with a control or miR-222 mimic oligonucleotide. 24 hours later, cells were pre-treated with an isotype control (IgG) or TNF neutralizing (α-TNF) antibody for two hours, and stimulated with 10 ng/ml LPS. Expression of the given genes was measured by qPCR (n=4 biologically independent samples). i , Efficacy of TNF neutralization was confirmed by treating cells with IgG or α-TNF as above, followed by stimulation with 100 ng/ml recombinant mouse TNF (n=3 biologically independent samples). Gene upregulation was not detected (ND) in 2/3 samples treated with α-TNF. For all bar graphs, mean +/− SEM is plotted. ** p

    Techniques Used: Sequencing, Binding Assay, Activity Assay, Luciferase, Construct, CRISPR, Clone Assay, Polymerase Chain Reaction, Expressing, Transfection, Selection, Real-time Polymerase Chain Reaction, Neutralization, Recombinant

    40) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Agarose gel analysis of DNA fragments generated during the MURA process. The Serratia sp. phospholipase gene (0.96 kb) was prepared by PCR (lane 1). The template DNA was digested with DNase I and the digests were purified (lane 2) and subjected to the MURA PCR as described in Materials and Methods; the resultant DNA fragments reassembled with a unidirectional primer (lane 3) were purified by using a Qiaquick purification kit. Lane M, 100-bp ladder DNA size marker.
    Figure Legend Snippet: Agarose gel analysis of DNA fragments generated during the MURA process. The Serratia sp. phospholipase gene (0.96 kb) was prepared by PCR (lane 1). The template DNA was digested with DNase I and the digests were purified (lane 2) and subjected to the MURA PCR as described in Materials and Methods; the resultant DNA fragments reassembled with a unidirectional primer (lane 3) were purified by using a Qiaquick purification kit. Lane M, 100-bp ladder DNA size marker.

    Techniques Used: Agarose Gel Electrophoresis, Generated, Polymerase Chain Reaction, Purification, Marker

    Related Articles

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    Article Snippet: .. Genomic DNA isolation kit, QIAquick® PCR purification kit, EpiTect®Bisulfite kit, and Effectene® transfection reagent were from Qiagen (Valencia, CA). .. MG132 was purchased from EMD Biosciences Inc. (San Diego, CA), and human genomic DNA was obtained from Clontech.

    Transfection:

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    Purification:

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination
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    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
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    Electrophoresis:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: .. Amplicons were confirmed by agarose electrophoresis, and purified with PCR purification kit (QIAGEN Cat.28104). .. 200 ng DNA samples were denatured and annealed according to the protocol in Ran et al . and subjected to T7E1 digestion in a 20 ul reaction according to the manufacturer’s protocol (NEB cat.M0302).

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    Incubation:

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins
    Article Snippet: .. In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104). ..

    Polymerase Chain Reaction:

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination
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    Article Snippet: .. In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104). ..

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    ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but <t>DNA</t> hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by <t>RT-PCR</t> (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).
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    ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

    Journal: BMC Biology

    Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

    doi: 10.1186/1741-7007-2-21

    Figure Lengend Snippet: ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

    Article Snippet: After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. (Valencia, CA).

    Techniques: Chromatin Immunoprecipitation, Modification, Methylation, Staining, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunoprecipitation

    Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated, Molecular Weight, Titration

    Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

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

    Journal: Epigenetics & Chromatin

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

    doi: 10.1186/s13072-018-0247-4

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

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

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

    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Journal: Scientific Reports

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    doi: 10.1038/s41598-020-58586-3

    Figure Lengend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Article Snippet: Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen).

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