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  • 99
    Qiagen minelute 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, 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, <t>MinElute</t> 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
    Minelute Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 16331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 16331 article reviews
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    99
    Thermo Fisher 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, 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, <t>MinElute</t> 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
    Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2092 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaquick pcr purification kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 135103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiagen minelute kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Qiagen Minelute Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen pipetman qiagen minelute 96 uf pcr purification kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Pipetman Qiagen Minelute 96 Uf Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pipetman qiagen minelute 96 uf pcr purification kit/product/Qiagen
    Average 99 stars, based on 7 article reviews
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    86
    Thermo Fisher qiagen purification kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Qiagen Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen mini plasmid purification kits
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Mini Plasmid Purification Kits, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen plasmid midi purification kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Plasmid Midi Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen plasmid maxi plasmid dna purification kit
    Real-time PCR analysis of viral <t>DNA</t> replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout <t>bacmid</t> or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.
    Plasmid Maxi Plasmid Dna Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen large construct dna purification kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Large Construct Dna Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen pyromark pcr kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Pyromark Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen longrange pcr kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Longrange Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen pcr cloningplus kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Pcr Cloningplus Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen minielute pcr purification kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Minielute Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1997 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/minielute pcr purification kit/product/Qiagen
    Average 99 stars, based on 1997 article reviews
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    99
    Qiagen type it pcr kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Type It Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen taq pcr core kit
    <t>WT-RANK</t> is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and <t>DNA</t> (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.
    Taq Pcr Core Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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: The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin.

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-020-58586-3

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0148023

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0148023

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0148023

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

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

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

    MORF2-REP profiles of Ethiopian T . evansi stocks and T . evansi and T . brucei reference strains. 1.5% agarose gel showing MORF2-REP minisatellite PCR amplicons. Lane M: 100 bp plus marker, lanes 1 to 14: Ethiopian T . evansi stocks MCAM/ET/2013/MU/01-02-04-05-06-07-08-09-10-11-13-14-15-17, lane 15: T . b . gambiense LiTat 1.3, lane 16: T . b . brucei AnTat 1.1 E lane 17: T . evansi type A (RoTat 1.2), lane 18: T . evansi type B (KETRI 2479), lane 19: T . equiperdum Dodola 940, lane 20: T . b . gambiense ABBA, lane N: negative control

    Journal: PLoS Neglected Tropical Diseases

    Article Title: New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels

    doi: 10.1371/journal.pntd.0004556

    Figure Lengend Snippet: MORF2-REP profiles of Ethiopian T . evansi stocks and T . evansi and T . brucei reference strains. 1.5% agarose gel showing MORF2-REP minisatellite PCR amplicons. Lane M: 100 bp plus marker, lanes 1 to 14: Ethiopian T . evansi stocks MCAM/ET/2013/MU/01-02-04-05-06-07-08-09-10-11-13-14-15-17, lane 15: T . b . gambiense LiTat 1.3, lane 16: T . b . brucei AnTat 1.1 E lane 17: T . evansi type A (RoTat 1.2), lane 18: T . evansi type B (KETRI 2479), lane 19: T . equiperdum Dodola 940, lane 20: T . b . gambiense ABBA, lane N: negative control

    Article Snippet: For direct sequencing, PCR was performed in 50–100 μl volumes and amplicons were cleaned up and concentrated using a PCR cleanup kit (QIAquick PCR Purification Kit, Qiagen, Germany) and sent out for bidirectional direct sequencing at the Genetic Sequencing Facility (VIB, Belgium) using the described PCR primers.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control

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

    Journal: Virology Journal

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

    doi: 10.1186/s12985-016-0504-8

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

    Article Snippet: Proteins were separated from the amplified DNA (“cleaned”) with either QIAquick PCR purification columns (Qiagen, Valencia, CA) or using one of the methods described below.

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

    Real-time PCR analysis of viral DNA replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout bacmid or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.

    Journal: Virology

    Article Title: Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame

    doi: 10.1016/j.virol.2007.05.003

    Figure Lengend Snippet: Real-time PCR analysis of viral DNA replication in transfected Sf-9 cells. Shown are the results of three independent DNA replication assays. For these analyses, total DNA was isolated from Sf-9 cells transfected with either the Ac 101 (panel A), 142 (panel B), or the 144 (panel C) knockout bacmid or a bacmid lacking the gp64 envelope fusion protein which served as the non-infectious control. At the designated time-point, total DNA was extracted and digested with the restriction enzyme Dpn I to eliminate input bacmid DNA, and analyzed by real-time PCR. The values displayed represent the averages from transfections performed in triplicate with error bars indicating standard deviations.

    Article Snippet: Bacmid DNA was purified from 0.5 L cultures using the plasmid Maxi plasmid DNA purification kit (Qiagen).

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Isolation, Knock-Out

    WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: WT-RANK is present in the ER, Golgi, and plasma membrane, but RANK mutants appear to accumulate intracellularly and are not detected predominantly in the Golgi or at the plasma membrane. Thus 293 cells transfected with ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG, ( C ) PDB-RANK-FLAG, and ( D ) ESH-RANK-FLAG were immunostained for FLAG ( green , i ) and stained for Golgi apparatus (wheat germ agglutinin-633; red , iii ), and DNA (Sytox green: blue , iii ) and analyzed by laser scanning confocal microscopy. xy sections of representative cells are presented. Panel iv in each case represents the merged images. Scale bar = 10 µm.

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Transfection, Staining, Confocal Microscopy

    Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: Mutant RANK accumulates intracellularly in human osteoclasts. Human osteoclasts transduced with adenoviral ( A ) WT-RANK-FLAG, ( B ) FEO-RANK-FLAG (including inset magnification of highlighted region), and ( C ) PDB-RANK-FLAG were immunostained for FLAG ( red ) and stained for DNA (Sytox green: green ) and analyzed by laser scanning confocal microscopy. Images presented are xy sections (scale bar shown on each image) of representative cells.

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Mutagenesis, Transduction, Staining, Confocal Microscopy

    Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Journal: Journal of Bone and Mineral Research

    Article Title: Signal peptide mutations in RANK prevent downstream activation of NF-?B

    doi: 10.1002/jbmr.399

    Figure Lengend Snippet: Confirmation of RANK expression in Flp-In cell lines by RTPCR, qPCR, and sequencing using construct-specific primers ( A ). The region around the tandem duplications in RANK was amplified by PCR 1 from genomic DNA from all Flp-In cell lines ( B , left panel ), and the products were sequenced and aligned to a consensus WT-RANK sequence to confirm that each cell line contained the expected duplication ( B , right panel : FEO 18 bp, PDB 27 bp, ESH 15 bp). The region around the FLAG tag was amplified by PCR 2 from genomic DNA from all Flp-In cell lines ( C , left panel , top ) with primers to amplify β-actin as housekeeping gene ( C , left panel , bottom ). The products were sequenced and aligned to a consensus RANK-FLAG sequence to confirm the presence of the C-terminal end of the RANK-FLAG constructs within each cell line ( C , right panel ). ( D ) PCR 3 was performed on genomic (g) and cDNA synthesized from RNA (c) extracted from all Flp-In cell lines, and PCR 4 was performed on genomic DNA ( E ). All PCR products were analyzed by ethidium bromide agarose gel electrophoresis with Hin cII DNA size markers, and the expected band sizes for each PCR reaction are shown in A . Data from QPCR (UPL probe 53) for ( F ) 293 cells that had been transiently transfected with RANK constructs or Flp-In cell lines stably expressing the RANK-FLAG genes and ( G ) 293, human peripheral blood mononuclear cells, and human osteoclast-like cells. The data represent the ΔΔ CP values normalized to GAPDH relative to expression levels in untransfected (UTX) cells. All qPCR data are representative of two independent experiments (three replicates per experiment). Note the log scale on the y axis in F .

    Article Snippet: The adenoviral DNA, containing the RANK cDNA, was purified using a large-construct DNA purification kit (Qiagen, Crawley, UK).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Construct, Amplification, Polymerase Chain Reaction, FLAG-tag, Synthesized, Agarose Gel Electrophoresis, Transfection, Stable Transfection