dsdna New England Biolabs Search Results


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  • 99
    New England Biolabs nebnext dsdna fragmentase
    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), <t>NEBNext</t> <t>dsDNA</t> <t>Fragmentase</t> (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.
    Nebnext Dsdna Fragmentase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher purelink 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, <t>PureLink®</t> 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
    Purelink Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polynucleotide kinase
    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, <t>PureLink®</t> 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
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher qubit dsdna hs assay 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, <t>PureLink®</t> 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
    Qubit Dsdna Hs Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 dna ligase
    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, <t>PureLink®</t> 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
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rq1 rnase free dnase
    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of <t>RNase</t> A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, <t>DNase</t> did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase
    Rq1 Rnase Free Dnase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 15967 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion high fidelity dna polymerase
    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of <t>RNase</t> A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, <t>DNase</t> did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs klenow fragment
    <t>UL30</t> inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM <t>Klenow</t> Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quant it picogreen dsdna assay kit
    <t>UL30</t> inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM <t>Klenow</t> Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.
    Quant It Picogreen Dsdna Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13403 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra dna library prep kit for illumina
    <t>UL30</t> inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM <t>Klenow</t> Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.
    Nebnext Ultra Dna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra ii dna library prep kit for illumina
    <t>UL30</t> inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM <t>Klenow</t> Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.
    Nebnext Ultra Ii Dna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1478 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ultra ii dna library prep kit
    Multiplex ligation-dependent probe amplification analysis of ectodermal dysplasia-associated genes. <t>DNA</t> was denatured and hybridized with SALSA probe mix, followed by ligation and polymerase chain reaction amplification. Capillary electrophoresis was performed to generate fragment length and peak area using GeneMapper version 3.0 software. Copy number ratio is denoted as the ratio of peak area of patient vs. peak area of references. Blue color peaks represent the patient sample, whereas the red color peaks were reference.
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna polymerase i
    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different <t>DNA</t> polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA <t>polymerase</t> I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs q5 high fidelity dna polymerase
    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different <t>DNA</t> polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA <t>polymerase</t> I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.
    Q5 High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna polymerase
    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with <t>T4</t> DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick pcr purification kit
    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with <t>T4</t> DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dapi
    ATF6β increases in mid-frontal cortical grey matter of patients with HAND and of patients infected with HIV. Formalin-fixed, paraffin-embedded mid-frontal cortical autopsy tissue was quadruple labelled with antibodies for ATF6β and for the cell-type markers for astrocytes (GFAP) and for neurones (MAP2), as well as the <t>DNA-binding</t> compound, <t>DAPI</t> to label nuclei. ( A ) Grey matter in labelled slides was imaged using laser confocal microscopy: ATF6β is shown in green, GFAP and MAP2 are shown in red and are not both present in the same images, and DAPI is shown in blue. Overlap of ATF6β with a cell-type marker appears yellow. ATF6β overlap with DAPI appears blue-green. An example is shown for an HIV(−) uninfected control, an HIV(+) neurocognitively normal case, and a HAND case for each cell-type marker. The third panel shows higher magnification images of astrocytes, which are denoted by asterisks [HIV(−) control] or arrowheads [HIV(+) cases]. ( B and C ) ATF6β integrated pixel intensity from confocal images (five per case) was quantified using Metamorph 6.0 software. Total ATF6β intensity increases in HAND cases ( n = 12) over neurocognitively normal (NcN) cases ( n = 5) ( B ) and in HIV(+) cases ( n = 14) over HIV(−) ( n = 3) controls ( C ). Values are shown as mean ± SEM. Student’s t -test and Mann–Whitney U post-hoc analysis was used for statistical analysis to determine significance. * P
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    Thermo Fisher m mlv reverse transcriptase
    ATF6β increases in mid-frontal cortical grey matter of patients with HAND and of patients infected with HIV. Formalin-fixed, paraffin-embedded mid-frontal cortical autopsy tissue was quadruple labelled with antibodies for ATF6β and for the cell-type markers for astrocytes (GFAP) and for neurones (MAP2), as well as the <t>DNA-binding</t> compound, <t>DAPI</t> to label nuclei. ( A ) Grey matter in labelled slides was imaged using laser confocal microscopy: ATF6β is shown in green, GFAP and MAP2 are shown in red and are not both present in the same images, and DAPI is shown in blue. Overlap of ATF6β with a cell-type marker appears yellow. ATF6β overlap with DAPI appears blue-green. An example is shown for an HIV(−) uninfected control, an HIV(+) neurocognitively normal case, and a HAND case for each cell-type marker. The third panel shows higher magnification images of astrocytes, which are denoted by asterisks [HIV(−) control] or arrowheads [HIV(+) cases]. ( B and C ) ATF6β integrated pixel intensity from confocal images (five per case) was quantified using Metamorph 6.0 software. Total ATF6β intensity increases in HAND cases ( n = 12) over neurocognitively normal (NcN) cases ( n = 5) ( B ) and in HIV(+) cases ( n = 14) over HIV(−) ( n = 3) controls ( C ). Values are shown as mean ± SEM. Student’s t -test and Mann–Whitney U post-hoc analysis was used for statistical analysis to determine significance. * P
    M Mlv Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher exonuclease i
    Proposed interpretations of mtDNA species detected by gel electrophoresis in  rnh 1 knockdown cells.  One of many possible scenarios is illustrated.  A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand.  B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR.  C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by  blue arrow  in   Fig. 6 C ).  D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products.  E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows  in   Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow  in   Fig. 6 B ).
    Exonuclease I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext multiplex oligos for illumina
    Proposed interpretations of mtDNA species detected by gel electrophoresis in  rnh 1 knockdown cells.  One of many possible scenarios is illustrated.  A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand.  B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR.  C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by  blue arrow  in   Fig. 6 C ).  D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products.  E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows  in   Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow  in   Fig. 6 B ).
    Nebnext Multiplex Oligos For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext end repair module
    Proposed interpretations of mtDNA species detected by gel electrophoresis in  rnh 1 knockdown cells.  One of many possible scenarios is illustrated.  A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand.  B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR.  C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by  blue arrow  in   Fig. 6 C ).  D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products.  E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows  in   Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow  in   Fig. 6 B ).
    Nebnext End Repair Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs exonuclease 1
    Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min <t>Exonuclease</t> 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.
    Exonuclease 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Journal: PLoS ONE

    Article Title: A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome

    doi: 10.1371/journal.pone.0112950

    Figure Lengend Snippet: Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Article Snippet: One µg of pUC19 plasmid DNA (Thermo Fisher, Erembodegem-Aalst, Belgium) was sheared by the Covaris or NEBNext dsDNA Fragmentase.

    Techniques: Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification

    ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using dsDNA fragmentase enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.

    Journal: Nature protocols

    Article Title: Generation of a transgenic ORFeome library in Drosophila

    doi: 10.1038/nprot.2014.105

    Figure Lengend Snippet: ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using dsDNA fragmentase enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.

    Article Snippet: For sequencing library preparation, a time course with the dsDNA fragmentase is useful for optimal digestion ( ).

    Techniques: Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Next-Generation Sequencing, Plasmid Preparation, Molecular Weight, Multiplexing

    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

    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of RNase A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, DNase did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase

    Journal: Human Molecular Genetics

    Article Title: Subcellular localization and RNAs determine FUS architecture in different cellular compartments

    doi: 10.1093/hmg/ddv239

    Figure Lengend Snippet: Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of RNase A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, DNase did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase

    Article Snippet: RNase-free DNase (catalog no. M6101) was purchased from Promega.

    Techniques:

    UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Article Snippet: AVAILABLE These data include: (1) the binding curve for NFAT DBD ( ); (2) effect of lac repressor on helicase DNA when the DNA lacks a lac repressor binding site ( ); (3) bound streptavidin does not affect DNA unwinding by the helicase ( ); (4) dissociation of streptavidin from duplex DNA in the presence of free biotin ( ); (5) effect of UL30 on Klenow Fragment activity ( ); (6) effect of streptavidin on DNA synthesis by UL30 and UL30-UL42 ( ); (7) specificity of lac repressor for DNA substrates containing the lac binding site , and; (8) comparison of the processivity of UL30 and Klenow Fragment ( ).

    Techniques: Agarose Gel Electrophoresis

    Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Article Snippet: AVAILABLE These data include: (1) the binding curve for NFAT DBD ( ); (2) effect of lac repressor on helicase DNA when the DNA lacks a lac repressor binding site ( ); (3) bound streptavidin does not affect DNA unwinding by the helicase ( ); (4) dissociation of streptavidin from duplex DNA in the presence of free biotin ( ); (5) effect of UL30 on Klenow Fragment activity ( ); (6) effect of streptavidin on DNA synthesis by UL30 and UL30-UL42 ( ); (7) specificity of lac repressor for DNA substrates containing the lac binding site , and; (8) comparison of the processivity of UL30 and Klenow Fragment ( ).

    Techniques: Agarose Gel Electrophoresis

    Multiplex ligation-dependent probe amplification analysis of ectodermal dysplasia-associated genes. DNA was denatured and hybridized with SALSA probe mix, followed by ligation and polymerase chain reaction amplification. Capillary electrophoresis was performed to generate fragment length and peak area using GeneMapper version 3.0 software. Copy number ratio is denoted as the ratio of peak area of patient vs. peak area of references. Blue color peaks represent the patient sample, whereas the red color peaks were reference.

    Journal: Molecular Medicine Reports

    Article Title: Gene screening facilitates diagnosis of complicated symptoms: A case report

    doi: 10.3892/mmr.2017.7590

    Figure Lengend Snippet: Multiplex ligation-dependent probe amplification analysis of ectodermal dysplasia-associated genes. DNA was denatured and hybridized with SALSA probe mix, followed by ligation and polymerase chain reaction amplification. Capillary electrophoresis was performed to generate fragment length and peak area using GeneMapper version 3.0 software. Copy number ratio is denoted as the ratio of peak area of patient vs. peak area of references. Blue color peaks represent the patient sample, whereas the red color peaks were reference.

    Article Snippet: PCR was performed with a primer cocktail using NEBNext® Ultra™ II DNA Library Prep kit (New England BioLabs, Inc.), followed by purification.

    Techniques: Multiplex Assay, Ligation, Amplification, Polymerase Chain Reaction, Electrophoresis, Software

    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Journal: Nucleic Acids Research

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    doi: 10.1093/nar/gkr424

    Figure Lengend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Article Snippet: After a 5-min incubation at 37°C, a 20 µl extension mix containing 66.8 mM Tris–HCl, 16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chloride, 62.5 U/ml T4 DNA Polymerase [or 62.5 U/ml Klenow fragment exo(−), 125 U/ml Klenow fragment, 125 U/ml DNA Polymerase I (Fermentas), 250 U/ml Exonuclease I (New England Biolabs)] was added.

    Techniques: Activity Assay, Hybridization, Generated

    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.

    Journal: Scientific Reports

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells

    doi: 10.1038/s41598-018-34580-8

    Figure Lengend Snippet: Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.

    Article Snippet: The sequencing adaptor oligos were: 5′-CCTCTCTATGGGCAGTCGGTGATTTTTTTT-3′ (universal adaptor) and 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG NNNNNNNNNN TTTTTTTT-3′ (barcoded adaptors, where NNNNNNNNNN represents an IonXpress barcode); End-repair II using T4 DNA polymerase (NEB) and dNTP mix (NEB).

    Techniques: Ligation, Sequencing, Hybridization, Two Tailed Test

    ATF6β increases in mid-frontal cortical grey matter of patients with HAND and of patients infected with HIV. Formalin-fixed, paraffin-embedded mid-frontal cortical autopsy tissue was quadruple labelled with antibodies for ATF6β and for the cell-type markers for astrocytes (GFAP) and for neurones (MAP2), as well as the DNA-binding compound, DAPI to label nuclei. ( A ) Grey matter in labelled slides was imaged using laser confocal microscopy: ATF6β is shown in green, GFAP and MAP2 are shown in red and are not both present in the same images, and DAPI is shown in blue. Overlap of ATF6β with a cell-type marker appears yellow. ATF6β overlap with DAPI appears blue-green. An example is shown for an HIV(−) uninfected control, an HIV(+) neurocognitively normal case, and a HAND case for each cell-type marker. The third panel shows higher magnification images of astrocytes, which are denoted by asterisks [HIV(−) control] or arrowheads [HIV(+) cases]. ( B and C ) ATF6β integrated pixel intensity from confocal images (five per case) was quantified using Metamorph 6.0 software. Total ATF6β intensity increases in HAND cases ( n = 12) over neurocognitively normal (NcN) cases ( n = 5) ( B ) and in HIV(+) cases ( n = 14) over HIV(−) ( n = 3) controls ( C ). Values are shown as mean ± SEM. Student’s t -test and Mann–Whitney U post-hoc analysis was used for statistical analysis to determine significance. * P

    Journal: Neuropathology and applied neurobiology

    Article Title: Activation status of integrated stress response pathways in neurones and astrocytes of HIV-associated neurocognitive disorders (HAND) cortex

    doi: 10.1111/j.1365-2990.2011.01215.x

    Figure Lengend Snippet: ATF6β increases in mid-frontal cortical grey matter of patients with HAND and of patients infected with HIV. Formalin-fixed, paraffin-embedded mid-frontal cortical autopsy tissue was quadruple labelled with antibodies for ATF6β and for the cell-type markers for astrocytes (GFAP) and for neurones (MAP2), as well as the DNA-binding compound, DAPI to label nuclei. ( A ) Grey matter in labelled slides was imaged using laser confocal microscopy: ATF6β is shown in green, GFAP and MAP2 are shown in red and are not both present in the same images, and DAPI is shown in blue. Overlap of ATF6β with a cell-type marker appears yellow. ATF6β overlap with DAPI appears blue-green. An example is shown for an HIV(−) uninfected control, an HIV(+) neurocognitively normal case, and a HAND case for each cell-type marker. The third panel shows higher magnification images of astrocytes, which are denoted by asterisks [HIV(−) control] or arrowheads [HIV(+) cases]. ( B and C ) ATF6β integrated pixel intensity from confocal images (five per case) was quantified using Metamorph 6.0 software. Total ATF6β intensity increases in HAND cases ( n = 12) over neurocognitively normal (NcN) cases ( n = 5) ( B ) and in HIV(+) cases ( n = 14) over HIV(−) ( n = 3) controls ( C ). Values are shown as mean ± SEM. Student’s t -test and Mann–Whitney U post-hoc analysis was used for statistical analysis to determine significance. * P

    Article Snippet: The GFAP and MAP2 primary antibodies were visualized by a Cy-5-conjugated, goat anti-rabbit antibody (1:200) and a Cy-3-conjugated goat anti-mouse antibody (1:200), respectively, and DAPI was used to visualize DNA (5 mM, Invitrogen).

    Techniques: Infection, Formalin-fixed Paraffin-Embedded, Binding Assay, Confocal Microscopy, Marker, Software, MANN-WHITNEY

    Proposed interpretations of mtDNA species detected by gel electrophoresis in  rnh 1 knockdown cells.  One of many possible scenarios is illustrated.  A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand.  B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR.  C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by  blue arrow  in   Fig. 6 C ).  D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products.  E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows  in   Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow  in   Fig. 6 B ).

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Proposed interpretations of mtDNA species detected by gel electrophoresis in rnh 1 knockdown cells. One of many possible scenarios is illustrated. A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand. B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR. C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by blue arrow in Fig. 6 C ). D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products. E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows in Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow in Fig. 6 B ).

    Article Snippet: For topology analysis, 1-μg aliquots of mitochondrial nucleic acid were incubated with the following enzymes, in 30 μl of manufacturer-supplied reaction buffer at 37 °C except where stated, and conditions as follows: topoisomerase I (New England Biolabs), 2 units, 30 min; gyrase (Topogen), 2 units, 60 min; restriction endonucleases MbiI, XhoI, EcoRV, NdeI, and Bsp1407I (Thermo Fisher Scientific), 4 units, 4 h; RNase H (Thermo Fisher Scientific), 0.5 unit, 60 min; S1 nuclease (Thermo Fisher Scientific), 2 units, 2 min at room temperature; RusA as described previously ( ); and exonuclease I (Thermo Fisher Scientific), 10 units, 60 min.

    Techniques: Nucleic Acid Electrophoresis, Derivative Assay

    Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.

    Journal: Communications Biology

    Article Title: Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

    doi: 10.1038/s42003-020-0768-9

    Figure Lengend Snippet: Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.

    Article Snippet: In vitro binding of the repair templates to the Cas9-VirD2 and VirD2-Cas9 fusion proteins For covalent binding of the Cas9-VirD2 and VirD2-Cas9 fusion proteins to the repair templates, the repair templates (1 µM of ssDNA or 300 ng of dsDNA) were incubated with 1 µg of either Cas9, Cas9-VirD, or VirD2-Cas9 at 37 °C for 30 min. For complete binding 1 µM of ssDNA or were incubated with 1 µg of Cas9-VirD at 37 °C in MgCl2 buffer for 5 min and 1 ul of Exonuclease 1 (E. coli , NEB) was added following manufacturer’s protocol and the reaction was incubated for 25 more minutes.

    Techniques: Expressing, Purification, SDS Page, Activity Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Binding Assay, Sequencing