polymerase chain reaction pcr clean up system  (Promega)

 
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    Name:
    Wizard PCR Preps DNA Purification System
    Description:
    Purifies PCR products directly from reactions in 15 minutes
    Catalog Number:
    a7170
    Price:
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    Category:
    Nucleic Acid Extraction Analysis Nucleic Acid Extraction Clean up and Concentration
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    Structured Review

    Promega polymerase chain reaction pcr clean up system
    The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter <t>nucleosome</t> repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by <t>qRT-PCR.</t> Expression levels were normalized to the maximum expression levels in wild-type (10 min).
    Purifies PCR products directly from reactions in 15 minutes
    https://www.bioz.com/result/polymerase chain reaction pcr clean up system/product/Promega
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    polymerase chain reaction pcr clean up system - by Bioz Stars, 2020-12
    99/100 stars

    Images

    1) Product Images from "INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes"

    Article Title: INO80 represses osmostress induced gene expression by resetting promoter proximal nucleosomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1292

    The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter nucleosome repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by qRT-PCR. Expression levels were normalized to the maximum expression levels in wild-type (10 min).
    Figure Legend Snippet: The H2A variant H2A.Z is not involved in INO80 related effects at stress genes. H2A.Z is not involved in delayed promoter nucleosome repopulation in arp8 Δ. ( A and B ) Nucleosome scanning assay of the CTT1 locus (−600 to +350 bp). Wild-type and htz1 Δ cells were treated with 0.4M NaCl for 10, 30 and 60 min or left untreated. NuSA are shown in (A) and quantification of NuSA of the CTT1 −1 and +1 nucleosomes in wild-type and htz1 Δ are shown in (B). Graphs in (B) are normalized to the untreated wild-type sample. ( C ) The stress induced transient recruitment of INO80 to the 5΄ end of the CTT1 locus is similar in wild-type and htz1 Δ mutant. Recruitment to the CTT1 5΄ region (+275) including the +1 nucleosome during osmotic stress (for indicated time points) was measured by ChIP of Ino80-TAP. ( D ) Expression of the CTT1 locus during osmotic stress is not influenced by absence of H2A.Z. Wild-type cells (BY4741), arp8 Δ, htz1 Δ and htz1 Δ arp8 Δ mutants were treated with 0.4M NaCl for indicated time points. CTT1 expression levels were quantified relative to VCX1 by qRT-PCR. Expression levels were normalized to the maximum expression levels in wild-type (10 min).

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

    Related Articles

    Amplification:

    Article Title: Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea
    Article Snippet: .. The amplified product from this PCR protocol was purified using the Wizard PCR Preps DNA purification system (Promega Corp., Madison, Wis.), and this purified preparation was used for DNA sequencing. .. The second PCR assessment utilized 6 sets of primers that bound the regions shown in Table .

    Article Title: MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR
    Article Snippet: .. After amplification, the resulting DNA fragments were extracted from PCR mixtures using Wizard PCR Preps DNA Purification System (Promega, Madison, USA). .. This purification procedure is required for separation of specific DNA fragments from primer dimers if they are formed during PCR.

    Agarose Gel Electrophoresis:

    Article Title: Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite Neotermes koshunensis
    Article Snippet: .. The PCR products were purified on a low-melting-point agarose gel by means of the Wizard PCR prep DNA purification system (Promega). .. The purified PCR products were digested with Hha I (Takara), and the lengths of the fluorescently labeled terminal restriction fragments of the PCR products were determined by electrophoresis by using an automatic sequencer, ALFred Express (Pharmacia), and analyzed by means of Fragment Manager software (Pharmacia).

    Ligation:

    Article Title: Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent
    Article Snippet: .. The ligation mixture was purified using the Wizard® PCR Preps DNA Purification Systems (Promega, USA) before the recombinant plasmids were transformed into FreeStyle™ CHO-S® cells. .. Expression of HN-acmA cassette

    Ethanol Precipitation:

    Article Title: More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli
    Article Snippet: .. The PCR product containing the kanamycin resistance cassette with flanking regions that are homologous to chromosomal sequences at the 5' and 3' end of the pdhR gene was purified with the Wizard DNA purification system (Promega), DpnI treated and further enriched by ethanol precipitation. .. In the next step it was transformed into BW25113 carrying pKD46.

    Purification:

    Article Title: Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea
    Article Snippet: .. The amplified product from this PCR protocol was purified using the Wizard PCR Preps DNA purification system (Promega Corp., Madison, Wis.), and this purified preparation was used for DNA sequencing. .. The second PCR assessment utilized 6 sets of primers that bound the regions shown in Table .

    Article Title: KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World
    Article Snippet: .. LSSP-PCR Analysis For the production of kDNA signatures from the 447 bp L. infantum kDNA fragments, PCR products were purified by using the kit Wizard® PCR Preps – DNA purification System (Promega). .. After 10 times dilution in deionized water, 2 µL of PCR-purified products were used as template in a second amplification step under low stringency conditions by using the MC1 primer as driver.

    Article Title: Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent
    Article Snippet: .. The ligation mixture was purified using the Wizard® PCR Preps DNA Purification Systems (Promega, USA) before the recombinant plasmids were transformed into FreeStyle™ CHO-S® cells. .. Expression of HN-acmA cassette

    Article Title: Production of a unique pneumococcal capsule serotype belonging to serogroup 6
    Article Snippet: .. PCR products were purified using Wizard PCR Clean-up System (Promega), and the DNA sequencing was performed by the genomics core facility at the University of Alabama at Birmingham (UAB). .. DNA sequences were analyzed with Lasergene v. 5.1 software (DNASTAR, Madison, WI) and were compared to the previously reported sequences of the 6B and 6C cps loci in GenBank (access numbers and , respectively) ( ).

    Article Title: More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli
    Article Snippet: .. The PCR product containing the kanamycin resistance cassette with flanking regions that are homologous to chromosomal sequences at the 5' and 3' end of the pdhR gene was purified with the Wizard DNA purification system (Promega), DpnI treated and further enriched by ethanol precipitation. .. In the next step it was transformed into BW25113 carrying pKD46.

    Article Title: Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite Neotermes koshunensis
    Article Snippet: .. The PCR products were purified on a low-melting-point agarose gel by means of the Wizard PCR prep DNA purification system (Promega). .. The purified PCR products were digested with Hha I (Takara), and the lengths of the fluorescently labeled terminal restriction fragments of the PCR products were determined by electrophoresis by using an automatic sequencer, ALFred Express (Pharmacia), and analyzed by means of Fragment Manager software (Pharmacia).

    DNA Sequencing:

    Article Title: Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea
    Article Snippet: .. The amplified product from this PCR protocol was purified using the Wizard PCR Preps DNA purification system (Promega Corp., Madison, Wis.), and this purified preparation was used for DNA sequencing. .. The second PCR assessment utilized 6 sets of primers that bound the regions shown in Table .

    Article Title: Production of a unique pneumococcal capsule serotype belonging to serogroup 6
    Article Snippet: .. PCR products were purified using Wizard PCR Clean-up System (Promega), and the DNA sequencing was performed by the genomics core facility at the University of Alabama at Birmingham (UAB). .. DNA sequences were analyzed with Lasergene v. 5.1 software (DNASTAR, Madison, WI) and were compared to the previously reported sequences of the 6B and 6C cps loci in GenBank (access numbers and , respectively) ( ).

    DNA Purification:

    Article Title: Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea
    Article Snippet: .. The amplified product from this PCR protocol was purified using the Wizard PCR Preps DNA purification system (Promega Corp., Madison, Wis.), and this purified preparation was used for DNA sequencing. .. The second PCR assessment utilized 6 sets of primers that bound the regions shown in Table .

    Article Title: Intramolecular Recombinations of Moloney Murine Leukemia Virus Occur during Minus-Strand DNA Synthesis
    Article Snippet: .. The remaining EDTA was removed by Wizard PCR DNA purification kit (Promega, Madison, Wis.). .. The proviruses of step 2 were amplified with primer GFP 2312 (CTGGAGTTCGTGACCG), which annealed within the 3′ end of gfp gene, and primer U3 3469 (GCTGGACCGCATCTGG), which annealed within the 3′ U3.

    Article Title: MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR
    Article Snippet: .. After amplification, the resulting DNA fragments were extracted from PCR mixtures using Wizard PCR Preps DNA Purification System (Promega, Madison, USA). .. This purification procedure is required for separation of specific DNA fragments from primer dimers if they are formed during PCR.

    Article Title: KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World
    Article Snippet: .. LSSP-PCR Analysis For the production of kDNA signatures from the 447 bp L. infantum kDNA fragments, PCR products were purified by using the kit Wizard® PCR Preps – DNA purification System (Promega). .. After 10 times dilution in deionized water, 2 µL of PCR-purified products were used as template in a second amplification step under low stringency conditions by using the MC1 primer as driver.

    Article Title: Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent
    Article Snippet: .. The ligation mixture was purified using the Wizard® PCR Preps DNA Purification Systems (Promega, USA) before the recombinant plasmids were transformed into FreeStyle™ CHO-S® cells. .. Expression of HN-acmA cassette

    Article Title: More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli
    Article Snippet: .. The PCR product containing the kanamycin resistance cassette with flanking regions that are homologous to chromosomal sequences at the 5' and 3' end of the pdhR gene was purified with the Wizard DNA purification system (Promega), DpnI treated and further enriched by ethanol precipitation. .. In the next step it was transformed into BW25113 carrying pKD46.

    Article Title: Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite Neotermes koshunensis
    Article Snippet: .. The PCR products were purified on a low-melting-point agarose gel by means of the Wizard PCR prep DNA purification system (Promega). .. The purified PCR products were digested with Hha I (Takara), and the lengths of the fluorescently labeled terminal restriction fragments of the PCR products were determined by electrophoresis by using an automatic sequencer, ALFred Express (Pharmacia), and analyzed by means of Fragment Manager software (Pharmacia).

    Polymerase Chain Reaction:

    Article Title: Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea
    Article Snippet: .. The amplified product from this PCR protocol was purified using the Wizard PCR Preps DNA purification system (Promega Corp., Madison, Wis.), and this purified preparation was used for DNA sequencing. .. The second PCR assessment utilized 6 sets of primers that bound the regions shown in Table .

    Article Title: Intramolecular Recombinations of Moloney Murine Leukemia Virus Occur during Minus-Strand DNA Synthesis
    Article Snippet: .. The remaining EDTA was removed by Wizard PCR DNA purification kit (Promega, Madison, Wis.). .. The proviruses of step 2 were amplified with primer GFP 2312 (CTGGAGTTCGTGACCG), which annealed within the 3′ end of gfp gene, and primer U3 3469 (GCTGGACCGCATCTGG), which annealed within the 3′ U3.

    Article Title: MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR
    Article Snippet: .. After amplification, the resulting DNA fragments were extracted from PCR mixtures using Wizard PCR Preps DNA Purification System (Promega, Madison, USA). .. This purification procedure is required for separation of specific DNA fragments from primer dimers if they are formed during PCR.

    Article Title: KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World
    Article Snippet: .. LSSP-PCR Analysis For the production of kDNA signatures from the 447 bp L. infantum kDNA fragments, PCR products were purified by using the kit Wizard® PCR Preps – DNA purification System (Promega). .. After 10 times dilution in deionized water, 2 µL of PCR-purified products were used as template in a second amplification step under low stringency conditions by using the MC1 primer as driver.

    Article Title: Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent
    Article Snippet: .. The ligation mixture was purified using the Wizard® PCR Preps DNA Purification Systems (Promega, USA) before the recombinant plasmids were transformed into FreeStyle™ CHO-S® cells. .. Expression of HN-acmA cassette

    Article Title: Production of a unique pneumococcal capsule serotype belonging to serogroup 6
    Article Snippet: .. PCR products were purified using Wizard PCR Clean-up System (Promega), and the DNA sequencing was performed by the genomics core facility at the University of Alabama at Birmingham (UAB). .. DNA sequences were analyzed with Lasergene v. 5.1 software (DNASTAR, Madison, WI) and were compared to the previously reported sequences of the 6B and 6C cps loci in GenBank (access numbers and , respectively) ( ).

    Article Title: More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli
    Article Snippet: .. The PCR product containing the kanamycin resistance cassette with flanking regions that are homologous to chromosomal sequences at the 5' and 3' end of the pdhR gene was purified with the Wizard DNA purification system (Promega), DpnI treated and further enriched by ethanol precipitation. .. In the next step it was transformed into BW25113 carrying pKD46.

    Article Title: Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite Neotermes koshunensis
    Article Snippet: .. The PCR products were purified on a low-melting-point agarose gel by means of the Wizard PCR prep DNA purification system (Promega). .. The purified PCR products were digested with Hha I (Takara), and the lengths of the fluorescently labeled terminal restriction fragments of the PCR products were determined by electrophoresis by using an automatic sequencer, ALFred Express (Pharmacia), and analyzed by means of Fragment Manager software (Pharmacia).

    Transformation Assay:

    Article Title: Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent
    Article Snippet: .. The ligation mixture was purified using the Wizard® PCR Preps DNA Purification Systems (Promega, USA) before the recombinant plasmids were transformed into FreeStyle™ CHO-S® cells. .. Expression of HN-acmA cassette

    Recombinant:

    Article Title: Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent
    Article Snippet: .. The ligation mixture was purified using the Wizard® PCR Preps DNA Purification Systems (Promega, USA) before the recombinant plasmids were transformed into FreeStyle™ CHO-S® cells. .. Expression of HN-acmA cassette

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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 362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wizard sv gel and pcr clean up system/product/Promega
    Average 99 stars, based on 362 article reviews
    Price from $9.99 to $1999.99
    wizard sv gel and pcr clean up system - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    99
    Promega wizard pcr clean up system
    <t>PCR</t> and <t>DNA</t> sequencing
    Wizard Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wizard pcr clean up system/product/Promega
    Average 99 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    wizard pcr clean up system - by Bioz Stars, 2020-12
    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

    High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

    Journal: The Journal of Cell Biology

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

    doi: 10.1083/jcb.201608083

    Figure Lengend Snippet: High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

    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.

    Techniques: Next-Generation Sequencing, Electrophoresis, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Sequencing, Affinity Purification

    CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

    Journal: The Journal of Cell Biology

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

    doi: 10.1083/jcb.201608083

    Figure Lengend Snippet: CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

    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.

    Techniques: In Vitro, Sedimentation, In Vivo, Expressing, Fractionation, Staining, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Differentiation of MRSA and MSSA isolates using conventional and normalized melt curve analysis. (a) Conventional and (b) normalized HRM curve analysis of PCR amplicons for CA-MRSA (ST93) (red), LA-MRSA (ST398) (blue), and MSSA (green) isolates.

    Journal: Journal of Clinical Microbiology

    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

    doi: 10.1128/JCM.02088-19

    Figure Lengend Snippet: Differentiation of MRSA and MSSA isolates using conventional and normalized melt curve analysis. (a) Conventional and (b) normalized HRM curve analysis of PCR amplicons for CA-MRSA (ST93) (red), LA-MRSA (ST398) (blue), and MSSA (green) isolates.

    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.

    Techniques: Polymerase Chain Reaction

    Conventional and normalized melt curve analysis of MRSA isolates. (a) Conventional and (b) normalized melt curve analysis of PCR amplicons from CA-MRSA (ST93) and LA-MRSA (ST398) isolates. All LA-MRSA isolates produced a single peak (mean 82.3°C), while CA-MRSA isolates produced one peak at higher temperature (mean 83.8°C) and a shoulder peak at lower temperature. The P071 (ST30) isolate was distinct from the ST93 and ST398 isolates.

    Journal: Journal of Clinical Microbiology

    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

    doi: 10.1128/JCM.02088-19

    Figure Lengend Snippet: Conventional and normalized melt curve analysis of MRSA isolates. (a) Conventional and (b) normalized melt curve analysis of PCR amplicons from CA-MRSA (ST93) and LA-MRSA (ST398) isolates. All LA-MRSA isolates produced a single peak (mean 82.3°C), while CA-MRSA isolates produced one peak at higher temperature (mean 83.8°C) and a shoulder peak at lower temperature. The P071 (ST30) isolate was distinct from the ST93 and ST398 isolates.

    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.

    Techniques: Polymerase Chain Reaction, Produced

    PCR and DNA sequencing

    Journal:

    Article Title: Production of a unique pneumococcal capsule serotype belonging to serogroup 6

    doi: 10.1099/mic.0.024521-0

    Figure Lengend Snippet: PCR and DNA sequencing

    Article Snippet: PCR products were purified using Wizard PCR Clean-up System (Promega), and the DNA sequencing was performed by the genomics core facility at the University of Alabama at Birmingham (UAB).

    Techniques: Polymerase Chain Reaction, DNA Sequencing