Structured Review

Promega pcr clean up system
Advantages and Experimental Procedure for Quantitative Abundance Profiling of the Microbiome in Plant Roots. (A) The major limitation of the current root microbiome profiling technique based on relative abundance is the lack of a method for quantitatively assessing the microbial load per unit amount of plant root tissue. (B) Three simulated root microbiomes associated with the same amounts of root tissue (upper panel) and the corresponding microbial profiling based on relative or quantitative abundance (lower panel). RA, relative abundance; QA, quantitative abundance, representing the copy-number ratio of microbial marker genes relative to plant genome. (C) Bar plots showing a comparison of relative (left panel) versus quantitative (right panel) microbial profiling of simulated root microbiome samples in (B) . (D) Schematic diagram showing the workflow of HA-QAP. (1) The artificial plasmid is designed and constructed as a spike-in control, containing conserved 16S rRNA, ITS primer regions (799F/1193R and ITS1F/ITS2), and a plant-specific gene sequence (the RID1 gene in this study). (2) <t>DNA</t> is extracted from the root microbiome, including plants and microbes. (3) A selected plant marker gene found in the total DNA is quantified by qPCR; this value is used to calibrate microbial relative abundance to quantitative abundance in step (7). (4) The predefined amount of spike-in plasmid is added to the DNA of root microbiome samples based on experience. (5 and 6) <t>PCR</t> amplicon libraries (bacteria and fungi, respectively) are prepared, sequenced (5), and analyzed (6). (7) The bacterial and fungal reads are calibrated based on the read counts of the spike-in and the amount of the plant marker gene quantified by qPCR in step (3) to determine the quantitative abundance relative to the host plant tissue.
Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Host-Associated Quantitative Abundance Profiling Reveals the Microbial Load Variation of Root Microbiome"

Article Title: Host-Associated Quantitative Abundance Profiling Reveals the Microbial Load Variation of Root Microbiome

Journal: Plant Communications

doi: 10.1016/j.xplc.2019.100003

Advantages and Experimental Procedure for Quantitative Abundance Profiling of the Microbiome in Plant Roots. (A) The major limitation of the current root microbiome profiling technique based on relative abundance is the lack of a method for quantitatively assessing the microbial load per unit amount of plant root tissue. (B) Three simulated root microbiomes associated with the same amounts of root tissue (upper panel) and the corresponding microbial profiling based on relative or quantitative abundance (lower panel). RA, relative abundance; QA, quantitative abundance, representing the copy-number ratio of microbial marker genes relative to plant genome. (C) Bar plots showing a comparison of relative (left panel) versus quantitative (right panel) microbial profiling of simulated root microbiome samples in (B) . (D) Schematic diagram showing the workflow of HA-QAP. (1) The artificial plasmid is designed and constructed as a spike-in control, containing conserved 16S rRNA, ITS primer regions (799F/1193R and ITS1F/ITS2), and a plant-specific gene sequence (the RID1 gene in this study). (2) DNA is extracted from the root microbiome, including plants and microbes. (3) A selected plant marker gene found in the total DNA is quantified by qPCR; this value is used to calibrate microbial relative abundance to quantitative abundance in step (7). (4) The predefined amount of spike-in plasmid is added to the DNA of root microbiome samples based on experience. (5 and 6) PCR amplicon libraries (bacteria and fungi, respectively) are prepared, sequenced (5), and analyzed (6). (7) The bacterial and fungal reads are calibrated based on the read counts of the spike-in and the amount of the plant marker gene quantified by qPCR in step (3) to determine the quantitative abundance relative to the host plant tissue.
Figure Legend Snippet: Advantages and Experimental Procedure for Quantitative Abundance Profiling of the Microbiome in Plant Roots. (A) The major limitation of the current root microbiome profiling technique based on relative abundance is the lack of a method for quantitatively assessing the microbial load per unit amount of plant root tissue. (B) Three simulated root microbiomes associated with the same amounts of root tissue (upper panel) and the corresponding microbial profiling based on relative or quantitative abundance (lower panel). RA, relative abundance; QA, quantitative abundance, representing the copy-number ratio of microbial marker genes relative to plant genome. (C) Bar plots showing a comparison of relative (left panel) versus quantitative (right panel) microbial profiling of simulated root microbiome samples in (B) . (D) Schematic diagram showing the workflow of HA-QAP. (1) The artificial plasmid is designed and constructed as a spike-in control, containing conserved 16S rRNA, ITS primer regions (799F/1193R and ITS1F/ITS2), and a plant-specific gene sequence (the RID1 gene in this study). (2) DNA is extracted from the root microbiome, including plants and microbes. (3) A selected plant marker gene found in the total DNA is quantified by qPCR; this value is used to calibrate microbial relative abundance to quantitative abundance in step (7). (4) The predefined amount of spike-in plasmid is added to the DNA of root microbiome samples based on experience. (5 and 6) PCR amplicon libraries (bacteria and fungi, respectively) are prepared, sequenced (5), and analyzed (6). (7) The bacterial and fungal reads are calibrated based on the read counts of the spike-in and the amount of the plant marker gene quantified by qPCR in step (3) to determine the quantitative abundance relative to the host plant tissue.

Techniques Used: Marker, Plasmid Preparation, Construct, Sequencing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

2) Product Images from "A rapid colorimetric LAMP assay for detection of Rhizoctonia solani AG-1 IA causing sheath blight of rice"

Article Title: A rapid colorimetric LAMP assay for detection of Rhizoctonia solani AG-1 IA causing sheath blight of rice

Journal: Scientific Reports

doi: 10.1038/s41598-020-79117-0

(A) PCR amplification using primer pair RSPG1Fand RSPG1R showing amplification product of 300 bp. (B) PCR amplification using primer pair RSPG2F and RSPG2R showing amplification product of 375 bp. (C) PCR amplification using primer pair RSPG4F and RSPG4R showing amplification product of 500 bp. (D) PCR amplification using primer pair RSPG5F and RSPG5R showing amplification product of 336 bp. Lane M is a 100-bp DNA marker. Lanes 1–51 represent different Rhizoctonia solani AG-1 IA strains; while lanes 52–58 are S. sclerotiorum , T. viride, F. oxyporum , A. alternata , C. capsici, B. subtilis, and P. plecoglossicida respectively. All DNA were extracted from fungal isolates.
Figure Legend Snippet: (A) PCR amplification using primer pair RSPG1Fand RSPG1R showing amplification product of 300 bp. (B) PCR amplification using primer pair RSPG2F and RSPG2R showing amplification product of 375 bp. (C) PCR amplification using primer pair RSPG4F and RSPG4R showing amplification product of 500 bp. (D) PCR amplification using primer pair RSPG5F and RSPG5R showing amplification product of 336 bp. Lane M is a 100-bp DNA marker. Lanes 1–51 represent different Rhizoctonia solani AG-1 IA strains; while lanes 52–58 are S. sclerotiorum , T. viride, F. oxyporum , A. alternata , C. capsici, B. subtilis, and P. plecoglossicida respectively. All DNA were extracted from fungal isolates.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

Related Articles

Clone Assay:

Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

Amplification:

Article Title: SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats 1
Article Snippet: .. Pooled whole-genome amplified DNA was purified by using the Wizard SV40 PCR Clean-up System (A9281; Promega). .. Purified DNA was checked on the gel and sent to Roche NimbleGen for ChIP-chip hybridization, and a three-plex promoter array was used for competitive hybridizations.

Mutagenesis:

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: .. Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282) .. 1 Set up the first PCR (AP-PCR #1) using 1 μl of genomic DNA, 1 μl of primers oPCR1 and Deg3 (10 μM each) using Taq .

Purification:

Article Title: SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats 1
Article Snippet: .. Pooled whole-genome amplified DNA was purified by using the Wizard SV40 PCR Clean-up System (A9281; Promega). .. Purified DNA was checked on the gel and sent to Roche NimbleGen for ChIP-chip hybridization, and a three-plex promoter array was used for competitive hybridizations.

Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega). .. Next-Generation Sequencing of the Coding Regions of mt Minichromosomes Purified PCR amplicons generated above with primers PLF1 and PLR from the coding regions of the mt minichromosomes of the domestic pig louse and the wild pig louse were sequenced initially with Roche GS FLX (454) platform at the AGRF and then with Illumina Hiseq 2000 platform at the Beijing Genomics Institute (BGI) for deeper coverages.

Article Title: Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis
Article Snippet: .. The PCR amplicons of selected samples were purified using the Wizard SV gel and PCR clean-up system (catalog no. A9281; Promega, Australia) following the manufacturer’s instructions. .. Purified amplicons were subjected to automated sequencing (Australian Genome Research Facility Ltd., Brisbane, Australia) in both directions, using the same primers used for PCR.

Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points
Article Snippet: .. DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit. .. The Bioanalyzer determines the quantity of DNA on the basis of fluorescence intensity.

Real-time Polymerase Chain Reaction:

Article Title: IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner
Article Snippet: .. DNA was finally eluted using the WIZARD® SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR). .. RNA immunoprecipitation (RIP) and quantitative RT-PCR analyses were performed essentially as recently described ( ).

Polymerase Chain Reaction:

Article Title: IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner
Article Snippet: .. DNA was finally eluted using the WIZARD® SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR). .. RNA immunoprecipitation (RIP) and quantitative RT-PCR analyses were performed essentially as recently described ( ).

Article Title: SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats 1
Article Snippet: .. Pooled whole-genome amplified DNA was purified by using the Wizard SV40 PCR Clean-up System (A9281; Promega). .. Purified DNA was checked on the gel and sent to Roche NimbleGen for ChIP-chip hybridization, and a three-plex promoter array was used for competitive hybridizations.

Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega). .. Next-Generation Sequencing of the Coding Regions of mt Minichromosomes Purified PCR amplicons generated above with primers PLF1 and PLR from the coding regions of the mt minichromosomes of the domestic pig louse and the wild pig louse were sequenced initially with Roche GS FLX (454) platform at the AGRF and then with Illumina Hiseq 2000 platform at the Beijing Genomics Institute (BGI) for deeper coverages.

Article Title: Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis
Article Snippet: .. The PCR amplicons of selected samples were purified using the Wizard SV gel and PCR clean-up system (catalog no. A9281; Promega, Australia) following the manufacturer’s instructions. .. Purified amplicons were subjected to automated sequencing (Australian Genome Research Facility Ltd., Brisbane, Australia) in both directions, using the same primers used for PCR.

Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
Article Snippet: .. Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282) .. 1 Set up the first PCR (AP-PCR #1) using 1 μl of genomic DNA, 1 μl of primers oPCR1 and Deg3 (10 μM each) using Taq .

DNA Purification:

Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points
Article Snippet: .. DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit. .. The Bioanalyzer determines the quantity of DNA on the basis of fluorescence intensity.

Sequencing:

Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega). .. Next-Generation Sequencing of the Coding Regions of mt Minichromosomes Purified PCR amplicons generated above with primers PLF1 and PLR from the coding regions of the mt minichromosomes of the domestic pig louse and the wild pig louse were sequenced initially with Roche GS FLX (454) platform at the AGRF and then with Illumina Hiseq 2000 platform at the Beijing Genomics Institute (BGI) for deeper coverages.

Plasmid Preparation:

Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

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    Promega wizard sv gel and pcr clean up system
    IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and <t>PCR</t> primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by <t>DNA</t> sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P
    Wizard Sv Gel And Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    Promega pcr clean up system
    Agarose gel electrophoresis of several <t>rbc</t> L <t>PCR</t> (A) and ITS2 fragment (B). Lane C shows negative control. The successful recombinant plasmid of PEasy- rbc L (C) and PEasy-ITS2 recombinant plasmid (D) is shown. 1 kb DNA ladder (Promega, Madison, WI).
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up system/product/Promega
    Average 99 stars, based on 3060 article reviews
    Price from $9.99 to $1999.99
    pcr clean up system - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

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    IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and PCR primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by DNA sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P

    Journal: Nucleic Acids Research

    Article Title: IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner

    doi: 10.1093/nar/gky1012

    Figure Lengend Snippet: IGF2BP1 promotes SRF expression in a 3′UTR and m 6 A-dependent manner. ( A ) Schematic depicting the position of IGF2BP1-CLIP sites reported in the SRF 3′UTR by six experiments performed in three indicated cell lines. ( B ) Schematic showing the SRF-3′UTR deletion strategy by CRISPR/Cas9. The relative position of sgRNAs and PCR primers for validating deletion of the SRF 3′UTR are indicated. ( C ) Representative semi-quantitative PCR analysis (left panel) of parental (WT) and SRF-3′UTR-deleted (Δ3′UTR) A549 cells. The successful deletion was further validated by DNA sequencing (right panel) of PCR products (Δ3′UTR) spanning the expected cleavage sites indicated in the schematic. ( D ) RT-qPCR analysis of indicated mRNAs in parental and SRF-Δ3′UTR A549 cells upon IGF2BP1 depletion (72 h). RPLP0 served as the normalization and GAPDH as the negative control. ( E ) Representative western blot analysis of indicated proteins in cells treated as described in (D). GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels upon IGF2BP1 depletion (relative to controls), as depicted above lower panel. ( F ) m 6 A -RIP-seq data showing m 6 ). ( G ) Representative western blot analysis of indicated proteins upon METTL3/14 depletion in parental (WT) and SRF-Δ3′UTR A549 cells. Note that IGF2BP1 expression is unaffected by METTL3/14 depletion, whereas SRF protein abundance is decreased only in parental A549 cells. GAPDH served as the loading and normalization control for the quantification ( n = 3) of SRF protein levels (relative to controls), as indicated in the lower panel. ( H ) The depletion of METTL3/14 by siRNA pools impairs SRF mRNA abundance in indicated cell lines. GAPDH served as the negative control in RT-qPCR studies cross-normalized to RPLP0 expression. ( I ) Altered m 6 A-modification of the SRF 3′UTR was determined upon METTL14 depletion in HepG2 cells by m 6 ). Note that the m 6 A-modified region partially overlaps with IGF2BP1-CLIP sites determined in HepG2 cells. ( J ) IGF2BP1-RIP analyses showing reduced association of the SRF mRNA with IGF2BP1 in METTL3/14-depleted A549 cells. RNA co-purified with IGF2BP1 from cells transfected with control siRNAs (siC) or METTL3/14-depleted cells was analyzed by RT-qPCR. HIST1H2AC served as the normalization and HIST2H3A as the negative control. Error bars indicate standard deviation determined in at least three analyses. Statistical significance was determined by Student’s t -test: (**) P

    Article Snippet: DNA was finally eluted using the WIZARD® SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR).

    Techniques: Expressing, Cross-linking Immunoprecipitation, CRISPR, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, DNA Sequencing, Quantitative RT-PCR, Negative Control, Western Blot, Modification, Purification, Transfection, Standard Deviation

    Agarose gel electrophoresis of several rbc L PCR (A) and ITS2 fragment (B). Lane C shows negative control. The successful recombinant plasmid of PEasy- rbc L (C) and PEasy-ITS2 recombinant plasmid (D) is shown. 1 kb DNA ladder (Promega, Madison, WI).

    Journal: Pharmaceutical Biology

    Article Title: Assessing product adulteration of Eurycoma longifolia (Tongkat Ali) herbal medicinal product using DNA barcoding and HPLC analysis

    doi: 10.1080/13880209.2018.1479869

    Figure Lengend Snippet: Agarose gel electrophoresis of several rbc L PCR (A) and ITS2 fragment (B). Lane C shows negative control. The successful recombinant plasmid of PEasy- rbc L (C) and PEasy-ITS2 recombinant plasmid (D) is shown. 1 kb DNA ladder (Promega, Madison, WI).

    Article Snippet: The amplified products of ITS2 and rbc L (FDB) were then purified using Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI) and ligated into pEasy-T3 cloning vector (Transgene Biotek, Hyderabad, Telangana) and transformed into an E. coli strain DH5α competent cells.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Recombinant, Plasmid Preparation

    H 2 O 2 production and release by cultured KGN cells and effects of exogenous H 2 O 2 . ( A ) NOX4 RT-PCR analysis and Western blot of cultured KGN cells show single bands of 160 bp and 68 kDa, respectively. Controls using RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Hydrogen peroxide assay of untreated KGN cells showed increasing H 2 O 2 levels in the supernatant over a time period of 2 h ( n = 6). Signal intensities were normalized to start point values. Bars indicate SEM. ( C ) Exogenously added H 2 O 2 reduced cell viability in a dose dependent manner. Cell counts after treatment of KGN cells with different concentrations of H 2 O 2 for 24 h ( n = 2–5 for each concentration) are shown with an interpolated sigmoidal standard curve ( r 2 = 0.9361). Bars indicate SEM. ( D ) Images of KGN cells treated with H 2 O 2 (1 mM) for 24 h compared to untreated control cells. Scale bars indicate 200 µm.

    Journal: Antioxidants

    Article Title: A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

    doi: 10.3390/antiox8110518

    Figure Lengend Snippet: H 2 O 2 production and release by cultured KGN cells and effects of exogenous H 2 O 2 . ( A ) NOX4 RT-PCR analysis and Western blot of cultured KGN cells show single bands of 160 bp and 68 kDa, respectively. Controls using RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Hydrogen peroxide assay of untreated KGN cells showed increasing H 2 O 2 levels in the supernatant over a time period of 2 h ( n = 6). Signal intensities were normalized to start point values. Bars indicate SEM. ( C ) Exogenously added H 2 O 2 reduced cell viability in a dose dependent manner. Cell counts after treatment of KGN cells with different concentrations of H 2 O 2 for 24 h ( n = 2–5 for each concentration) are shown with an interpolated sigmoidal standard curve ( r 2 = 0.9361). Bars indicate SEM. ( D ) Images of KGN cells treated with H 2 O 2 (1 mM) for 24 h compared to untreated control cells. Scale bars indicate 200 µm.

    Article Snippet: Amplicon identity was verified by agarose gel electrophoresis, consecutive cDNA extraction with Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) and sequence analysis (GATC, Konstanz, Germany).

    Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, H2O2 Assay, Concentration Assay

    KGN cells express functional TRPM2. ( A ) TRPM2 RT-PCR shows a band at 103 bp. Controls with RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Addition of H 2 O 2 (1 mM) increased the fluorescence signal of the four individual KGN cells shown, which were loaded with the Ca 2+ -sensitive dye Fluoforte. Background signals were subtracted and fluorescence is shown relative to the respective start value of each region of interest (ROI). ( C ) Fluorescence images, taken before ( a ) and after ( b ) the first stimulation with H 2 O 2 . ( D ) Treatment with the inhibitor (ACA; 20 µM), 4 h prior to and during the measurement, blocked the Ca 2+ increase upon stimulation with H 2 O 2 , but not with 0.05‰ trypsin (T). Images ( c – f ) represent the indicated time points. ( E ) The H 2 O 2 -derived Ca 2+ increase was obtained in the DMSO control and thus ruled out solvent effects. Images ( g – j ) represent the indicated time points. The pseudo-color scale shown in ( c ) applies for all live cell images. Colored frames mark the cells represented in the corresponding graphs. Scale bars indicate 50 µm.

    Journal: Antioxidants

    Article Title: A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

    doi: 10.3390/antiox8110518

    Figure Lengend Snippet: KGN cells express functional TRPM2. ( A ) TRPM2 RT-PCR shows a band at 103 bp. Controls with RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Addition of H 2 O 2 (1 mM) increased the fluorescence signal of the four individual KGN cells shown, which were loaded with the Ca 2+ -sensitive dye Fluoforte. Background signals were subtracted and fluorescence is shown relative to the respective start value of each region of interest (ROI). ( C ) Fluorescence images, taken before ( a ) and after ( b ) the first stimulation with H 2 O 2 . ( D ) Treatment with the inhibitor (ACA; 20 µM), 4 h prior to and during the measurement, blocked the Ca 2+ increase upon stimulation with H 2 O 2 , but not with 0.05‰ trypsin (T). Images ( c – f ) represent the indicated time points. ( E ) The H 2 O 2 -derived Ca 2+ increase was obtained in the DMSO control and thus ruled out solvent effects. Images ( g – j ) represent the indicated time points. The pseudo-color scale shown in ( c ) applies for all live cell images. Colored frames mark the cells represented in the corresponding graphs. Scale bars indicate 50 µm.

    Article Snippet: Amplicon identity was verified by agarose gel electrophoresis, consecutive cDNA extraction with Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) and sequence analysis (GATC, Konstanz, Germany).

    Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Derivative Assay

    Cay1 and Rap1 genetically interact to maintain telomere homeostasis A Telomere length analysis of Apa I-digested DNA from cay1 Δ trt1 Δ cells harvested at increasing generation doublings (gen). B Telomere length analysis of Apa I-digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. C PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1-arrested by nitrogen starvation (G1). Genomic DNA was digested with Not I and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). D Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. E qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t -test. ** P

    Journal: The EMBO Journal

    Article Title: Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels

    doi: 10.15252/embj.201489559

    Figure Lengend Snippet: Cay1 and Rap1 genetically interact to maintain telomere homeostasis A Telomere length analysis of Apa I-digested DNA from cay1 Δ trt1 Δ cells harvested at increasing generation doublings (gen). B Telomere length analysis of Apa I-digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. C PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1-arrested by nitrogen starvation (G1). Genomic DNA was digested with Not I and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). D Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. E qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t -test. ** P

    Article Snippet: Immunoprecipitated chromatin was eluted in elution buffer (1% SDS, 100 mM sodium bicarbonate, 40 mg/ml RNase A) and incubated at 37°C for 1 h. Cross-links were reversed at 65°C for 16 h and DNA purified with the Wizard SV Gel and PCR Clean-Up System (Promega).

    Techniques: Northern Blot, Quantitative RT-PCR, Two Tailed Test