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    Structured Review

    Thermo Fisher pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit <t>mRNA</t> abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Pcr Clean Up Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up kit/product/Thermo Fisher
    Average 97 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice"

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13193-3

    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Figure Legend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Techniques Used: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test

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    Amplification:

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    Article Snippet: .. The DNA and cDNA for each sample were amplified by WGA separately, and excess random primers were removed using a ChargeSwitch-Pro PCR cleanup kit (Invitrogen, Carlsbad, CA, USA). .. The cleaned WGA DNA and cDNA products were quantified on a Nanodrop to obtain equal concentrations (ng · μl−1 ) of DNA and cDNA in the 24 recombined samples.

    Article Title: Distinct Gut Virome Profile of Pregnant Women With Type 1 Diabetes in the ENDIA Study
    Article Snippet: .. After amplification, polymerase chain reaction (PCR) products were visualized on an agarose gel before purification using the ChargeSwitch-Pro PCR CleanUp Kit (Thermo Fisher Scientific). .. Virome Capture Sequencing One microgram of double-stranded DNA (dsDNA) was used for library synthesis using the KAPA Hyperplus kit (KAPA Biosystems, Wilmington, MA) with single-index adapters.

    Whole Genome Amplification:

    Article Title: Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream
    Article Snippet: .. The DNA and cDNA for each sample were amplified by WGA separately, and excess random primers were removed using a ChargeSwitch-Pro PCR cleanup kit (Invitrogen, Carlsbad, CA, USA). .. The cleaned WGA DNA and cDNA products were quantified on a Nanodrop to obtain equal concentrations (ng · μl−1 ) of DNA and cDNA in the 24 recombined samples.

    Purification:

    Article Title: Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
    Article Snippet: .. The PCR product > 500 bp was excised and purified from the gel using Wizard® SV Gel and PCR Clean-Up System as per kit’s instruction. .. The purified PCR product was sent to NovogeneAIT ( https://en.novogene.com/ ) for sequencing.

    Article Title: Haplotypes of CYP1B1 and CCDC57 genes in an Afro-Caribbean female population with uterine leiomyoma.
    Article Snippet: .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry.

    Article Title: Diversity of retrievable heterotrophic bacteria in Kongsfjorden, an Arctic fjord
    Article Snippet: .. The amplicons were purified using ChargeSwitch Pro PCR cleanup kit (Invitrogen, Carlsbad, CA, USA). .. The eluted fragments were sequenced using an automated DNA sequencing system (Applied Biosystems, Carlsbad, CA, USA).

    Article Title: Distinct Gut Virome Profile of Pregnant Women With Type 1 Diabetes in the ENDIA Study
    Article Snippet: .. After amplification, polymerase chain reaction (PCR) products were visualized on an agarose gel before purification using the ChargeSwitch-Pro PCR CleanUp Kit (Thermo Fisher Scientific). .. Virome Capture Sequencing One microgram of double-stranded DNA (dsDNA) was used for library synthesis using the KAPA Hyperplus kit (KAPA Biosystems, Wilmington, MA) with single-index adapters.

    Article Title: Oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus in Northern Greece
    Article Snippet: .. PCR products were purified using the Chargeswitch PCR cleanup (Invitrogen, UK) according to the manufacturer's instructions. .. Purified PCR products were sequenced at an ABI 310 platform using the appropriate primer pair for each amplified product.

    Article Title: Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina
    Article Snippet: .. PCR products were run on an agarose gel to verify the predicted product sizes and purified using the ChargeSwitch-Pro PCR clean-up kit (Invitrogen). ..

    Polymerase Chain Reaction:

    Article Title: Removing auto-activators from yeast-two-hybrid assays by conditional negative selection
    Article Snippet: .. The PCR product > 500 bp was excised and purified from the gel using Wizard® SV Gel and PCR Clean-Up System as per kit’s instruction. .. The purified PCR product was sent to NovogeneAIT ( https://en.novogene.com/ ) for sequencing.

    Article Title: Novel 5-Nitrofuran-Activating Reductase in Escherichia coli
    Article Snippet: .. The ahpF amplicons were cleaned up using ChargeSwitch-Pro PCR Clean-Up kit (Invitrogen) according to the manufacturer’s instructions and submitted to the Massey Genome Service (Massey University, Palmerston North, New Zealand) for DNA Sanger sequencing using BigDye Terminator v3.1. .. The primers used for sequencing included ahpF forward primer, ahpF reverse primer, and ahpF internal forward primer (5′-GTTCACCTCGCTGGTACTGG-3′).

    Article Title: Haplotypes of CYP1B1 and CCDC57 genes in an Afro-Caribbean female population with uterine leiomyoma.
    Article Snippet: .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry.

    Article Title: Diversity of retrievable heterotrophic bacteria in Kongsfjorden, an Arctic fjord
    Article Snippet: .. The amplicons were purified using ChargeSwitch Pro PCR cleanup kit (Invitrogen, Carlsbad, CA, USA). .. The eluted fragments were sequenced using an automated DNA sequencing system (Applied Biosystems, Carlsbad, CA, USA).

    Article Title: Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream
    Article Snippet: .. The DNA and cDNA for each sample were amplified by WGA separately, and excess random primers were removed using a ChargeSwitch-Pro PCR cleanup kit (Invitrogen, Carlsbad, CA, USA). .. The cleaned WGA DNA and cDNA products were quantified on a Nanodrop to obtain equal concentrations (ng · μl−1 ) of DNA and cDNA in the 24 recombined samples.

    Article Title: Distinct Gut Virome Profile of Pregnant Women With Type 1 Diabetes in the ENDIA Study
    Article Snippet: .. After amplification, polymerase chain reaction (PCR) products were visualized on an agarose gel before purification using the ChargeSwitch-Pro PCR CleanUp Kit (Thermo Fisher Scientific). .. Virome Capture Sequencing One microgram of double-stranded DNA (dsDNA) was used for library synthesis using the KAPA Hyperplus kit (KAPA Biosystems, Wilmington, MA) with single-index adapters.

    Article Title: Oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus in Northern Greece
    Article Snippet: .. PCR products were purified using the Chargeswitch PCR cleanup (Invitrogen, UK) according to the manufacturer's instructions. .. Purified PCR products were sequenced at an ABI 310 platform using the appropriate primer pair for each amplified product.

    Article Title: Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina
    Article Snippet: .. PCR products were run on an agarose gel to verify the predicted product sizes and purified using the ChargeSwitch-Pro PCR clean-up kit (Invitrogen). ..

    Generated:

    Article Title: Haplotypes of CYP1B1 and CCDC57 genes in an Afro-Caribbean female population with uterine leiomyoma.
    Article Snippet: .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry.

    Sequencing:

    Article Title: Novel 5-Nitrofuran-Activating Reductase in Escherichia coli
    Article Snippet: .. The ahpF amplicons were cleaned up using ChargeSwitch-Pro PCR Clean-Up kit (Invitrogen) according to the manufacturer’s instructions and submitted to the Massey Genome Service (Massey University, Palmerston North, New Zealand) for DNA Sanger sequencing using BigDye Terminator v3.1. .. The primers used for sequencing included ahpF forward primer, ahpF reverse primer, and ahpF internal forward primer (5′-GTTCACCTCGCTGGTACTGG-3′).

    Article Title: Haplotypes of CYP1B1 and CCDC57 genes in an Afro-Caribbean female population with uterine leiomyoma.
    Article Snippet: .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. .. Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry.

    Agarose Gel Electrophoresis:

    Article Title: Distinct Gut Virome Profile of Pregnant Women With Type 1 Diabetes in the ENDIA Study
    Article Snippet: .. After amplification, polymerase chain reaction (PCR) products were visualized on an agarose gel before purification using the ChargeSwitch-Pro PCR CleanUp Kit (Thermo Fisher Scientific). .. Virome Capture Sequencing One microgram of double-stranded DNA (dsDNA) was used for library synthesis using the KAPA Hyperplus kit (KAPA Biosystems, Wilmington, MA) with single-index adapters.

    Article Title: Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina
    Article Snippet: .. PCR products were run on an agarose gel to verify the predicted product sizes and purified using the ChargeSwitch-Pro PCR clean-up kit (Invitrogen). ..

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    Thermo Fisher genejet pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, <t>GeneJET</t> PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Genejet Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quantitative analysis of RNA <t>transcription</t> by qRT-PCR. <t>In</t> <t>vitro</t> transcription was performed, and the resulting RNA was reverse transcribed into cDNA, followed by qRT-PCR quantification of the cDNA. (A) The template DNA was serially diluted 10-fold for seven times and subjected to qRT-PCR. The cycle thresholds (CTs) were plotted against the expected copy numbers of the template DNA, showing a linear relationship (Y = −3.677x + 40.12, r 2 = 0.9628). This standard curve was then used for the calculation of RNA copy number generated from transcription reactions. The graph represents the combined results of three independent experiments. Error bars represent standard deviation. (B) The RNA copy numbers generated from transcription were plotted against the amounts of HeLa nuclear extract used in the reactions, followed by fitting with the Michaelis-Menten model and the non-linear least-squares method in Excel spreadsheets (Y = 9.568*X/(11.34+X) - 0.01128*X - 0.02921, r 2 = 0.9773). Substitution of HeLa nuclear extract with 5 μg of C. elegans nuclear extract in the transcription reaction produced 102,589 RNA molecules on average. The graph represents the combined results of three independent experiments. Error bars represent standard deviation.
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    Thermo Fisher pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit <t>mRNA</t> abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Pcr Clean Up Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    pcr clean up kit - by Bioz Stars, 2020-09
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    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

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

    doi: 10.1186/s12864-017-4371-5

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

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

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

    TAZ interacts with c-Jun for Irs1 transcription. a Mouse gastrocnemius muscle genomic DNA was sheared by sonication, and chromatin fragments were immunoprecipitated with c-Jun antibodies. ChIPed DNA was analysed by qRT-PCR with primer sets flanking TBE1 ( n = 3). Eight- to ten-week-old mice were used. b TAZ interacted with c-Jun. 293T cells were co-transfected with F-TAZ- and c-Jun-expressing plasmids. After immunoprecipitating with anti-FLAG antibodies, the eluted samples were analysed by immunoblotting with TAZ and c-Jun antibodies. c Endogenous c-Jun interacted with TAZ. C2C12 cell lysates were immunoprecipitated with IgG or c-Jun antibodies, and immune complexes were detected using TAZ and c-Jun antibodies. d Mutating c-Jun-binding sites in TBE1-luc decreased TAZ- and c-Jun-mediated reporter activity. The mutant c-Jun-binding site was generated by site-directed mutagenesis. Luciferase reporter plasmids harbouring wild-type (TBE-luc) or mutant c-Jun-binding sites (TBE1m-luc) were transfected into 293T cells together with c-Jun- and TAZ-expressing plasmids. Luciferase reporter gene activity was analysed 24 h after transfection ( n = 3). e Wild-type (amino acids 1–395), N-terminus-deleted TAZ (amino acids 124–395 and 164–395) and WW domain-truncated TAZ expression plasmids (ΔWW) were transfected into 293T cells together with a c-Jun expression plasmid. Cell lysates were immunoprecipitated 24 h after transfection with FLAG antibodies, and the eluted samples were analysed by immunoblotting using TAZ and c-Jun antibodies. f The transcriptional activity of the TAZ deletion mutant was assessed using a TBE1-luc reporter plasmid. 293T cells were co-transfected with the TAZ deletion mutant together with a c-Jun expression plasmid as described in e . A Renilla luciferase plasmid was used for transfection normalisation. Cells were lysed, and luciferase activity was measured 24 h after transfection ( n = 3). Data are presented as mean ± SD. Statistical analysis was performed using Student’s t -test. ns not significant; *** p

    Journal: Nature Communications

    Article Title: TAZ couples Hippo/Wnt signalling and insulin sensitivity through Irs1 expression

    doi: 10.1038/s41467-019-08287-x

    Figure Lengend Snippet: TAZ interacts with c-Jun for Irs1 transcription. a Mouse gastrocnemius muscle genomic DNA was sheared by sonication, and chromatin fragments were immunoprecipitated with c-Jun antibodies. ChIPed DNA was analysed by qRT-PCR with primer sets flanking TBE1 ( n = 3). Eight- to ten-week-old mice were used. b TAZ interacted with c-Jun. 293T cells were co-transfected with F-TAZ- and c-Jun-expressing plasmids. After immunoprecipitating with anti-FLAG antibodies, the eluted samples were analysed by immunoblotting with TAZ and c-Jun antibodies. c Endogenous c-Jun interacted with TAZ. C2C12 cell lysates were immunoprecipitated with IgG or c-Jun antibodies, and immune complexes were detected using TAZ and c-Jun antibodies. d Mutating c-Jun-binding sites in TBE1-luc decreased TAZ- and c-Jun-mediated reporter activity. The mutant c-Jun-binding site was generated by site-directed mutagenesis. Luciferase reporter plasmids harbouring wild-type (TBE-luc) or mutant c-Jun-binding sites (TBE1m-luc) were transfected into 293T cells together with c-Jun- and TAZ-expressing plasmids. Luciferase reporter gene activity was analysed 24 h after transfection ( n = 3). e Wild-type (amino acids 1–395), N-terminus-deleted TAZ (amino acids 124–395 and 164–395) and WW domain-truncated TAZ expression plasmids (ΔWW) were transfected into 293T cells together with a c-Jun expression plasmid. Cell lysates were immunoprecipitated 24 h after transfection with FLAG antibodies, and the eluted samples were analysed by immunoblotting using TAZ and c-Jun antibodies. f The transcriptional activity of the TAZ deletion mutant was assessed using a TBE1-luc reporter plasmid. 293T cells were co-transfected with the TAZ deletion mutant together with a c-Jun expression plasmid as described in e . A Renilla luciferase plasmid was used for transfection normalisation. Cells were lysed, and luciferase activity was measured 24 h after transfection ( n = 3). Data are presented as mean ± SD. Statistical analysis was performed using Student’s t -test. ns not significant; *** p

    Article Snippet: DNA samples were purified using a Gel Extraction/PCR Purification Kit (Thermo).

    Techniques: Sonication, Immunoprecipitation, Quantitative RT-PCR, Mouse Assay, Transfection, Expressing, Binding Assay, Activity Assay, Mutagenesis, Generated, Luciferase, Plasmid Preparation

    TAZ stimulates Irs1 transcription with c-Jun and Tead4. a Chromatin immunoprecipitation (ChIP) sequencing analysis was assessed in FLAG-tagged TAZ (F-TAZ)-overexpressing or control C2C12 myoblasts. Sites of F-TAZ binding were visualised in the mouse genome. The primary target site for TAZ-binding (TAZ-binding element 1, TBE1) is marked with an asterisk. The histone modification status (H3K27ac, H3K4 me3, and H3K4 me1) was obtained from raw ChIP sequencing read data downloaded from the NCBI Sequence Read Archive (SRP009088). b Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) was performed to validate the TAZ-binding element 1 (TBE1) site. ChIP was performed in control and F-TAZ-overexpressing C2C12 cells. ChIPed DNA was analysed by qPCR with primer sets spanning the TBE1 site ( n = 3). c Scrambled or two different siRNAs for both c-Jun and Tead4 were co-transfected into C2C12 cells. Transfected cells were analysed by qRT-PCR for Irs1 transcripts 48 h after transfection ( n = 3). d Depleting c-Jun and Tead4 decreased IRS1 level. C2C12 cells were treated with two different siRNAs for c-Jun and Tead4 and lysates were analysed by immunoblotting. e TAZ and c-Jun stimulate a TBE1-containing luciferase reporter gene. Approximately 500 bp of intronic elements surrounding TBE1 were cloned into the pGL3-basic luciferase vector (TBE1-luc), and the completed plasmid was transfected into 293T cells together with c-Jun-, Tead4-, and/or TAZ-expressing plasmids. Luciferase activity was analysed 24 h after transfection. The pGL3-basic vector was used as a control ( n = 3). f TAZ stimulated TBE1-driven gene transcription. Control and TAZ-knockdown 293T cells (Ti) were transfected with pGL3-basic or TBE1-luc, and a luciferase assay was performed 24 h after transfection ( n = 3). Data are presented as mean ± SD. Statistical analysis was performed using a Student’s t -test. *** p

    Journal: Nature Communications

    Article Title: TAZ couples Hippo/Wnt signalling and insulin sensitivity through Irs1 expression

    doi: 10.1038/s41467-019-08287-x

    Figure Lengend Snippet: TAZ stimulates Irs1 transcription with c-Jun and Tead4. a Chromatin immunoprecipitation (ChIP) sequencing analysis was assessed in FLAG-tagged TAZ (F-TAZ)-overexpressing or control C2C12 myoblasts. Sites of F-TAZ binding were visualised in the mouse genome. The primary target site for TAZ-binding (TAZ-binding element 1, TBE1) is marked with an asterisk. The histone modification status (H3K27ac, H3K4 me3, and H3K4 me1) was obtained from raw ChIP sequencing read data downloaded from the NCBI Sequence Read Archive (SRP009088). b Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) was performed to validate the TAZ-binding element 1 (TBE1) site. ChIP was performed in control and F-TAZ-overexpressing C2C12 cells. ChIPed DNA was analysed by qPCR with primer sets spanning the TBE1 site ( n = 3). c Scrambled or two different siRNAs for both c-Jun and Tead4 were co-transfected into C2C12 cells. Transfected cells were analysed by qRT-PCR for Irs1 transcripts 48 h after transfection ( n = 3). d Depleting c-Jun and Tead4 decreased IRS1 level. C2C12 cells were treated with two different siRNAs for c-Jun and Tead4 and lysates were analysed by immunoblotting. e TAZ and c-Jun stimulate a TBE1-containing luciferase reporter gene. Approximately 500 bp of intronic elements surrounding TBE1 were cloned into the pGL3-basic luciferase vector (TBE1-luc), and the completed plasmid was transfected into 293T cells together with c-Jun-, Tead4-, and/or TAZ-expressing plasmids. Luciferase activity was analysed 24 h after transfection. The pGL3-basic vector was used as a control ( n = 3). f TAZ stimulated TBE1-driven gene transcription. Control and TAZ-knockdown 293T cells (Ti) were transfected with pGL3-basic or TBE1-luc, and a luciferase assay was performed 24 h after transfection ( n = 3). Data are presented as mean ± SD. Statistical analysis was performed using a Student’s t -test. *** p

    Article Snippet: DNA samples were purified using a Gel Extraction/PCR Purification Kit (Thermo).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Binding Assay, Modification, Real-time Polymerase Chain Reaction, Transfection, Quantitative RT-PCR, Luciferase, Clone Assay, Plasmid Preparation, Expressing, Activity Assay

    Quantitative analysis of RNA transcription by qRT-PCR. In vitro transcription was performed, and the resulting RNA was reverse transcribed into cDNA, followed by qRT-PCR quantification of the cDNA. (A) The template DNA was serially diluted 10-fold for seven times and subjected to qRT-PCR. The cycle thresholds (CTs) were plotted against the expected copy numbers of the template DNA, showing a linear relationship (Y = −3.677x + 40.12, r 2 = 0.9628). This standard curve was then used for the calculation of RNA copy number generated from transcription reactions. The graph represents the combined results of three independent experiments. Error bars represent standard deviation. (B) The RNA copy numbers generated from transcription were plotted against the amounts of HeLa nuclear extract used in the reactions, followed by fitting with the Michaelis-Menten model and the non-linear least-squares method in Excel spreadsheets (Y = 9.568*X/(11.34+X) - 0.01128*X - 0.02921, r 2 = 0.9773). Substitution of HeLa nuclear extract with 5 μg of C. elegans nuclear extract in the transcription reaction produced 102,589 RNA molecules on average. The graph represents the combined results of three independent experiments. Error bars represent standard deviation.

    Journal: bioRxiv

    Article Title: A novel in vitro Caenorhabditis elegans transcription system

    doi: 10.1101/2020.06.22.149609

    Figure Lengend Snippet: Quantitative analysis of RNA transcription by qRT-PCR. In vitro transcription was performed, and the resulting RNA was reverse transcribed into cDNA, followed by qRT-PCR quantification of the cDNA. (A) The template DNA was serially diluted 10-fold for seven times and subjected to qRT-PCR. The cycle thresholds (CTs) were plotted against the expected copy numbers of the template DNA, showing a linear relationship (Y = −3.677x + 40.12, r 2 = 0.9628). This standard curve was then used for the calculation of RNA copy number generated from transcription reactions. The graph represents the combined results of three independent experiments. Error bars represent standard deviation. (B) The RNA copy numbers generated from transcription were plotted against the amounts of HeLa nuclear extract used in the reactions, followed by fitting with the Michaelis-Menten model and the non-linear least-squares method in Excel spreadsheets (Y = 9.568*X/(11.34+X) - 0.01128*X - 0.02921, r 2 = 0.9773). Substitution of HeLa nuclear extract with 5 μg of C. elegans nuclear extract in the transcription reaction produced 102,589 RNA molecules on average. The graph represents the combined results of three independent experiments. Error bars represent standard deviation.

    Article Snippet: Titration of HeLa nuclear extract standards was performed using the in vitro transcription assay followed by qRT-PCR quantification.

    Techniques: Quantitative RT-PCR, In Vitro, Generated, Standard Deviation, Produced

    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Journal: Nature Communications

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    doi: 10.1038/s41467-019-13193-3

    Figure Lengend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Article Snippet: Cas9 mRNA (Addgene #42251) was generated by linearization with PmeI, purified with PCR clean-up kit, and transcribed with mMESSAGE mMACHINE T7 Kit (Thermo Fisher Scientific).

    Techniques: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test