deoxynucleotide triphosphates New England Biolabs Search Results


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  • 99
    New England Biolabs t4 pnk
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dntp mix
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
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    New England Biolabs dntp mix
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Dntp Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e coli dna ligase
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    E Coli Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dnase i
    IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either <t>DNase</t> I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8732 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs escherichia coli dna polymerase i
    IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either <t>DNase</t> I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P
    Escherichia Coli Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs alu i
    <t>Alu</t> I digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).
    Alu I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 842 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase
    <t>Alu</t> I digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).
    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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    New England Biolabs ecop15i
    Overview of the method (A) Schematic outline of the method. The method starts with the construction of a 3C library. The library is then digested with a second restriction enzyme (RE2) and followed by DNA circularization. The resulting circular DNA is digested again with the primary 3C RE1. The re-opened RE1 sites are ligated to an adapter containing an <t>EcoP15I</t> restriction site and marked with biotin through DNA circularization, and the DNA circles are cut by the enzyme EcoP15I to produce biotin-labeled paired-end tags of 25-27 bp, which are isolated, PCR amplified and finally sequenced on a next generation sequencing platform. (B) Schematic of the structure of the DNA molecules containing the paired-end tags in the chromatin interaction library constructed using our method.
    Ecop15i, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nt bbvci
    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. <t>SacI</t> cuts both strands of mtDNA once (linear), Nt. <t>BbvCI</t> cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    Nt Bbvci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 rna ligase 1
    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. <t>SacI</t> cuts both strands of mtDNA once (linear), Nt. <t>BbvCI</t> cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    T4 Rna Ligase 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dgtp
    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. <t>SacI</t> cuts both strands of mtDNA once (linear), Nt. <t>BbvCI</t> cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
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    New England Biolabs dpnii
    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. <t>SacI</t> cuts both strands of mtDNA once (linear), Nt. <t>BbvCI</t> cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
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    New England Biolabs t5 exonuclease
    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 10 u psti hf
    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
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    New England Biolabs t4 rna ligase 2
    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
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    New England Biolabs bsai
    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
    Bsai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bsmi
    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
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    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Journal: bioRxiv

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    doi: 10.1101/2020.01.22.915009

    Figure Lengend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Article Snippet: End-repair with T4 DNA Polymerase: for the treatments +/− and +/+, an end-repair mastermix was made by combining 4 μl T4 DNA Ligase Reaction Buffer (NEB), 0.5 μl dNTP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (25% PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl), 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl), 1.5 μl T4 DNA Polymerase (NEB, cat#M0203S, 3 U/μl) and 0.1 μl Taq DNA Polymerase (NEB, cat#M0273S, 5 U/μl).

    Techniques: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P

    Journal: Infection and Immunity

    Article Title: Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-?B ▿

    doi: 10.1128/IAI.01317-07

    Figure Lengend Snippet: IL-8 secretion induced by conditioned supernatants generated from Campylobacter -T84 cell coculture. (A) Polarized T84 cells were incubated for 24 h either with bacterium-free conditioned supernatants generated from the apical or basolateral supernatant of polarized T84 cells that had been inoculated with C. jejuni 81-176 for 4 h or with C. jejuni 81-176 alone cultured in the invasion medium for 4 h (bacterial). T84 cells were incubated with the bacterial culture medium in the apical chamber, with the apical conditioned supernatant in the apical chamber, or with the basolateral conditioned supernatant in the basolateral chamber. Polarized T84 cells inoculated apically with live C. jejuni 81-176 for 4 h and 24 h were used as controls. (B) The basolateral conditioned supernatant generated from C. jejuni 81-176-T84 cell coculture either was not pretreated (−) or was pretreated with either DNase I (10 U/ml) at 37°C for 2 h, polymyxin B (20 μg/ml) at 37°C for 30 min (PLXB), protease K (100 μg/ml) overnight followed by a 20-min incubation at 100°C (ProtK), or a 20-min incubation at 100°C without protease K (Boiling). Polarized T84 cells were incubated with the pretreated or untreated conditioned supernatants from wt C. jejuni 81-176-T84 cell coculture in the basolateral chamber at 37°C for 24 h. (C) Polarized T84 cells were treated basolaterally with a C. jejuni 81-176 DNA extract (25 μg/ml) (DNA) and DNase I-treated C. jejuni 81-176 DNA (DNA + DNase) at 37°C for 24 h. (D) Polarized T84 cells were incubated basolaterally with different concentrations of E. coli LPS in the presence or absence of 20 μg/ml PLXB. (E) Polarized T84 cells were incubated with conditioned supernatants from a coculture of T84 cells with wt C. jejuni 81-176 or its flaA , pflA , cdtB , or flaA cdtB mutant in the basolateral chamber at 37°C for 24 h. After different treatments, the apical (filled bars) and basolateral (open bars) media were collected. IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. The data in panels B and E are expressed as the ratio of the IL-8 level induced by a treated basolateral conditioned supernatant to that induced by an untreated supernatant and as the ratio of the IL-8 level induced by a mutant basolateral conditioned supernatant to that induced by a wt supernatant. *, P

    Article Snippet: To inactivate heat-sensitive proteins, the collected conditioned supernatant was boiled for 20 min. To remove most of the protein factors, the conditioned supernatant was treated with protease K (100 μg/ml; Invitrogen) at 56°C overnight, followed by a 20-min incubation at 100°C to inactivate protease K. To remove bacterial DNA, the conditioned supernatant was treated with DNase I (10 U/ml; New England Biolabs, Ipswich, MA) at 37°C for 2 h. The treated supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and agarose gel electrophoresis, confirming the removal of proteins by protease K and of bacterial DNA by DNase I (data not shown).

    Techniques: Generated, Incubation, Cell Culture, Mutagenesis, Enzyme-linked Immunosorbent Assay

    Alu I digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).

    Journal: Journal of Virological Methods

    Article Title: Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

    doi: 10.1016/j.jviromet.2004.07.008

    Figure Lengend Snippet: Alu I digestion of amplicons from SARS-HCoV, HCoV-229E and HCoV-OC43. Lanes 1, 3 and 5 (MW): molecular weight markers (Amplisize ladder 50–2000 bp, Bio-Rad Laboratories, Mississauga, Canada); Lane 2: SARS-HCoV (predicted fragments: 126 bp, 94 bp); Lane 4: HCoV-229E (predicted fragments: 101 bp, 86 bp, 33 bp); Lane 6: HCoV-OC43 (predicted fragment: 220 bp).

    Article Snippet: The digestion reaction consisted of 10 μl of the RT-PCR mixture, 1.5 μl of the 10× enzyme buffer, 1 μl of Alu I (10 U/μl, New England Biolabs) and 2.5 μl of ddH2 O for a total of 15 μl.

    Techniques: Molecular Weight

    Overview of the method (A) Schematic outline of the method. The method starts with the construction of a 3C library. The library is then digested with a second restriction enzyme (RE2) and followed by DNA circularization. The resulting circular DNA is digested again with the primary 3C RE1. The re-opened RE1 sites are ligated to an adapter containing an EcoP15I restriction site and marked with biotin through DNA circularization, and the DNA circles are cut by the enzyme EcoP15I to produce biotin-labeled paired-end tags of 25-27 bp, which are isolated, PCR amplified and finally sequenced on a next generation sequencing platform. (B) Schematic of the structure of the DNA molecules containing the paired-end tags in the chromatin interaction library constructed using our method.

    Journal: Methods (San Diego, Calif.)

    Article Title: A genome-wide 3C-method for characterizing the three-dimensional architectures of genomes

    doi: 10.1016/j.ymeth.2012.06.018

    Figure Lengend Snippet: Overview of the method (A) Schematic outline of the method. The method starts with the construction of a 3C library. The library is then digested with a second restriction enzyme (RE2) and followed by DNA circularization. The resulting circular DNA is digested again with the primary 3C RE1. The re-opened RE1 sites are ligated to an adapter containing an EcoP15I restriction site and marked with biotin through DNA circularization, and the DNA circles are cut by the enzyme EcoP15I to produce biotin-labeled paired-end tags of 25-27 bp, which are isolated, PCR amplified and finally sequenced on a next generation sequencing platform. (B) Schematic of the structure of the DNA molecules containing the paired-end tags in the chromatin interaction library constructed using our method.

    Article Snippet: Experimental steps: 33 Add 15 μl 10x NEBuffer 3, 1.5 μl BSA, 2.5 μl EcoP15I (10 u/ μl, NEB), 1.8 μl S-adenosylmethionine (final concentration = 380 μM), and 50 μl water to 80 μl RE1 re-linearized DNA (2-3 μg), and incubate overnight at 37°C.

    Techniques: Labeling, Isolation, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing, Construct

    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Journal: Nucleic Acids Research

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    doi: 10.1093/nar/gky456

    Figure Lengend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Article Snippet: To visualize different topological isomers of the mtDNA, 400 ng of control total DNA was additionally incubated at 37°C for 30 min with only buffer (no treatment), SacI (linear) (New England Biolabs; 20 U), Nt.BbvCI (nicked circles) (New England Biolabs; 10 U), Topo I (relaxes the closed circles) (New England Biolabs; 5 U), DNA gyrase (compacts the closed circles) (New England Biolabs; 5 U).

    Techniques: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Journal: BMC Biotechnology

    Article Title: Patch cloning method for multiple site-directed and saturation mutagenesis

    doi: 10.1186/1472-6750-13-91

    Figure Lengend Snippet: Schematic illustration of the multiple patch cloning procedure.  DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Article Snippet: The MUPAC enzyme stock solution was prepared by mixing 10 μL of T5 exonuclease (10 U/μL; New England Biolabs), 1 μL of Klenow fragment exo– (5 U/μL; New England Biolabs), 2.5 μL of T4 DNA ligase (400 U/μL; New England Biolabs), and 11.5 μL of the storage buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, and 0.1% Triton X-100).

    Techniques: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation