datp  (New England Biolabs)


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    dATP Solution
    Description:
    dATP Solution 25 umol
    Catalog Number:
    N0440S
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    54
    Category:
    Deoxynucleotides
    Size:
    25 umol
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    New England Biolabs datp
    dATP Solution
    dATP Solution 25 umol
    https://www.bioz.com/result/datp/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    Images

    1) Product Images from "Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation"

    Article Title: Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1134

    Specificity of M. jannaschii PSTK for the phosphoryl donor. Initial velocities for the phosphotransferase activity of PSTK (6 nM PSTK without NTP or the addition of ATP, ITP, GTP, CTP, UTP or dATP or 200 nM PSTK with AMP-CPP or AMP-PCP) in the presence of the indicated phosphoryl donors (20 mM). Reactions proceeded for 6 min at 37°C and 1.5 min time points were taken and analyzed as in Figure 1 A and 2 . *ND, no detectable phosphorylation. Error bars represent the standard deviation of three experiments.
    Figure Legend Snippet: Specificity of M. jannaschii PSTK for the phosphoryl donor. Initial velocities for the phosphotransferase activity of PSTK (6 nM PSTK without NTP or the addition of ATP, ITP, GTP, CTP, UTP or dATP or 200 nM PSTK with AMP-CPP or AMP-PCP) in the presence of the indicated phosphoryl donors (20 mM). Reactions proceeded for 6 min at 37°C and 1.5 min time points were taken and analyzed as in Figure 1 A and 2 . *ND, no detectable phosphorylation. Error bars represent the standard deviation of three experiments.

    Techniques Used: Activity Assay, Conditioned Place Preference, Standard Deviation

    2) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    Figure Legend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Techniques Used: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.
    Figure Legend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Techniques Used: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    3) Product Images from "Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase"

    Article Title: Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm416

    Simulated systems after 2 ns MD. Color code: light-pink, protein (ribbon representation); magenta, Tyr-714; grey, template DNA strand; cyan, primer DNA strand; orange, Mg 2+ ions (sphere representation); yellow, modified dG*; red, BP moiety; blue, dATP/dA; green, dCTP/dC; light blue, dGTP/dG; chocolate, dTTP/dT. ( k–m ) are the three simulated systems with [BP]G* at the post-insertion site of open BF: two computer-generated models ( k,1 ) and one crystal structure ( m ). The arrows in ( s ) indicate the motion of the Watson-Crick edges of modified guanine and partner C. The arrow in ( v ) indicates the movement of the phosphate of [BP]G*. Figure 4 is continued on the next page.
    Figure Legend Snippet: Simulated systems after 2 ns MD. Color code: light-pink, protein (ribbon representation); magenta, Tyr-714; grey, template DNA strand; cyan, primer DNA strand; orange, Mg 2+ ions (sphere representation); yellow, modified dG*; red, BP moiety; blue, dATP/dA; green, dCTP/dC; light blue, dGTP/dG; chocolate, dTTP/dT. ( k–m ) are the three simulated systems with [BP]G* at the post-insertion site of open BF: two computer-generated models ( k,1 ) and one crystal structure ( m ). The arrows in ( s ) indicate the motion of the Watson-Crick edges of modified guanine and partner C. The arrow in ( v ) indicates the movement of the phosphate of [BP]G*. Figure 4 is continued on the next page.

    Techniques Used: Modification, Generated, Polyacrylamide Gel Electrophoresis

    ( a ) Template-primer strand duplex used in the running start experiment showing the numbering scheme. ( b ) Typical running start primer extension experiments catalyzed by BF as a function of incubation time (min). Extension of the primer strand, a 22-mer with the terminal 3′-G base opposite the template base C labeled ‘–3’ on the template strand. The position of the [BP]G* is labeled ‘0’ (25-mer). The ‘–1’ position denotes a 24-mer. The lane marked ‘M’ contains 22-mer, 24-mer, 25-mer and 43-mer primer strands (the 43-mer corresponds to the length of a fully extended primer strand). Reactions were initiated with 4.5 nM of the primer/template duplex (1:1.5 ratio), 20 nM of BF, and 200 μM of dNTPs at 37°C. ( c ) Typical single step 2′-deoxynucleotide triphosphate (dNTP) insertion catalyzed by BF. The primer strand was 24 nucleotides long with the terminal 3′-C positioned opposite the template base labeled ‘–1’ which corresponds to the darkest bands in this figure. Incorporation of each individual dNTP opposite the [BP]G* adduct results in the lighter bands just above the darkest bands, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and Ctr = ‘–1’ and ‘0’ position marker. Reactions were initiated at 2 mM of each dNTP, 15 nM of [DNA], and 2 nM of BF at 37°C for 30 min.
    Figure Legend Snippet: ( a ) Template-primer strand duplex used in the running start experiment showing the numbering scheme. ( b ) Typical running start primer extension experiments catalyzed by BF as a function of incubation time (min). Extension of the primer strand, a 22-mer with the terminal 3′-G base opposite the template base C labeled ‘–3’ on the template strand. The position of the [BP]G* is labeled ‘0’ (25-mer). The ‘–1’ position denotes a 24-mer. The lane marked ‘M’ contains 22-mer, 24-mer, 25-mer and 43-mer primer strands (the 43-mer corresponds to the length of a fully extended primer strand). Reactions were initiated with 4.5 nM of the primer/template duplex (1:1.5 ratio), 20 nM of BF, and 200 μM of dNTPs at 37°C. ( c ) Typical single step 2′-deoxynucleotide triphosphate (dNTP) insertion catalyzed by BF. The primer strand was 24 nucleotides long with the terminal 3′-C positioned opposite the template base labeled ‘–1’ which corresponds to the darkest bands in this figure. Incorporation of each individual dNTP opposite the [BP]G* adduct results in the lighter bands just above the darkest bands, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and Ctr = ‘–1’ and ‘0’ position marker. Reactions were initiated at 2 mM of each dNTP, 15 nM of [DNA], and 2 nM of BF at 37°C for 30 min.

    Techniques Used: Incubation, Labeling, Marker

    4) Product Images from "Replisome-mediated translesion synthesis by a cellular replicase"

    Article Title: Replisome-mediated translesion synthesis by a cellular replicase

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.800441

    The stalled leading strand is extended directly by TLS. A , pulse-chase, EcoRI runoff replication reactions were performed either in the absence or presence of elevated concentrations of dATP and dCTP using the CPD template as described under “Experimental procedures.” In these reactions, replication is initiated in the absence of DNA gyrase and allowed to proceed for 1 min when the replication forks stall because of the accumulation of positive supercoils. The template is then digested with EcoRI in less than 10 s at the same time as [α- 32 P]dGTP is added. After 1 min, the radiolabel is chased with a 100-fold excess of cold nucleotide. Time points are as indicated postchase. The products were analyzed by electrophoresis through either denaturing alkaline agarose gels ( top ) or native agarose gels ( bottom ). B , lane traces of the 1-, 3-, and 6-min lanes in the presence of elevated dATP and dCTP in A (denaturing). FL , full length; PSL , photostimulated luminescence.
    Figure Legend Snippet: The stalled leading strand is extended directly by TLS. A , pulse-chase, EcoRI runoff replication reactions were performed either in the absence or presence of elevated concentrations of dATP and dCTP using the CPD template as described under “Experimental procedures.” In these reactions, replication is initiated in the absence of DNA gyrase and allowed to proceed for 1 min when the replication forks stall because of the accumulation of positive supercoils. The template is then digested with EcoRI in less than 10 s at the same time as [α- 32 P]dGTP is added. After 1 min, the radiolabel is chased with a 100-fold excess of cold nucleotide. Time points are as indicated postchase. The products were analyzed by electrophoresis through either denaturing alkaline agarose gels ( top ) or native agarose gels ( bottom ). B , lane traces of the 1-, 3-, and 6-min lanes in the presence of elevated dATP and dCTP in A (denaturing). FL , full length; PSL , photostimulated luminescence.

    Techniques Used: Pulse Chase, Electrophoresis

    Lesion bypass is directly proportional to nucleotide concentration. A , standard replication reactions with the indicated total concentrations of dATP ( dA ) and dCTP ( dC ) were analyzed by denaturing agarose gel electrophoresis. B , quantification of the results shown in A . Shown is the mean from two experiments. FL , full length.
    Figure Legend Snippet: Lesion bypass is directly proportional to nucleotide concentration. A , standard replication reactions with the indicated total concentrations of dATP ( dA ) and dCTP ( dC ) were analyzed by denaturing agarose gel electrophoresis. B , quantification of the results shown in A . Shown is the mean from two experiments. FL , full length.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis

    5) Product Images from "Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase"

    Article Title: Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx633

    Fluorometric assay to detect RNA-dependent Hs PrimPol DNA primase/polymerase activity. ( A ) Fluorescence kinetics as a measure of de novo DNA synthesis by purified Hs PrimPol (450 nM), in combination with different components as indicated. An increase in fluorescence as a function of time occurred only when dATP and dGTP (100 μM each), the GTCC template (1 μM; see Supplementary Table S1 ), and MnCl 2 (1 mM) were added. The use of the ATCC template (1 μM; Supplementary Table S1 ), or MgCl 2 as metal donor, did not render a measurable fluorescence signal. ( B ) Fluorescence kinetics using the optimal conditions described in A, but comparing GTCC and GUCC templates (see Supplementary Table S1 ). A negative control assay using GTCC template in the absence of dNTPs is represented as a comparison. Note that the scale of arbitrary units in the y-axis is different with respect to A. ( C ) Representative electropherogram of a priming experiment carried out with purified Hs PrimPol (450 nM) and 16 nM [γ-32P]ATP, 100 μM dGTP and either GTCC or GUCC as template. The position of the labeled ‘5′-AdG-3′ dimer is indicated. Detailed experimental conditions, as well as templates sequences are described in Materials and Methods.
    Figure Legend Snippet: Fluorometric assay to detect RNA-dependent Hs PrimPol DNA primase/polymerase activity. ( A ) Fluorescence kinetics as a measure of de novo DNA synthesis by purified Hs PrimPol (450 nM), in combination with different components as indicated. An increase in fluorescence as a function of time occurred only when dATP and dGTP (100 μM each), the GTCC template (1 μM; see Supplementary Table S1 ), and MnCl 2 (1 mM) were added. The use of the ATCC template (1 μM; Supplementary Table S1 ), or MgCl 2 as metal donor, did not render a measurable fluorescence signal. ( B ) Fluorescence kinetics using the optimal conditions described in A, but comparing GTCC and GUCC templates (see Supplementary Table S1 ). A negative control assay using GTCC template in the absence of dNTPs is represented as a comparison. Note that the scale of arbitrary units in the y-axis is different with respect to A. ( C ) Representative electropherogram of a priming experiment carried out with purified Hs PrimPol (450 nM) and 16 nM [γ-32P]ATP, 100 μM dGTP and either GTCC or GUCC as template. The position of the labeled ‘5′-AdG-3′ dimer is indicated. Detailed experimental conditions, as well as templates sequences are described in Materials and Methods.

    Techniques Used: Activity Assay, Fluorescence, DNA Synthesis, Purification, Negative Control, Labeling

    RNA-dependent DNA polymerase activity shown by WT HsPrimPol and Y89R variant. ( A ) Names and sequences of oligonucleotides used as template/primer hybrids to measure primer-dependent dNTP incorporation by Hs PrimPol; RNA sequences are indicated in italics; ‘*’ indicates the radiolabeled oligonucleotide used as primer. ( B ) Polymerization reactions by WT or Y89R variant Hs PrimPol was evaluated using the indicated combinations of dNTPs (100 μM each) and either DNA (T13T/Spc1) or RNA ( T13U /Spc1) templates. As shown by electrophoretic analysis, primers (15-mer) elongated in 2, 4, 6 or 13 residues represent the expected products in the presence of either dATP, dATP+dGTP, dATP+dGTP+dTTP, or the four dNTPs, respectively.
    Figure Legend Snippet: RNA-dependent DNA polymerase activity shown by WT HsPrimPol and Y89R variant. ( A ) Names and sequences of oligonucleotides used as template/primer hybrids to measure primer-dependent dNTP incorporation by Hs PrimPol; RNA sequences are indicated in italics; ‘*’ indicates the radiolabeled oligonucleotide used as primer. ( B ) Polymerization reactions by WT or Y89R variant Hs PrimPol was evaluated using the indicated combinations of dNTPs (100 μM each) and either DNA (T13T/Spc1) or RNA ( T13U /Spc1) templates. As shown by electrophoretic analysis, primers (15-mer) elongated in 2, 4, 6 or 13 residues represent the expected products in the presence of either dATP, dATP+dGTP, dATP+dGTP+dTTP, or the four dNTPs, respectively.

    Techniques Used: Activity Assay, Variant Assay

    6) Product Images from "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing"

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    Journal: bioRxiv

    doi: 10.1101/2020.06.23.166397

    Comparison of different Bst polymerases for RT-LAMP. A) Performance of Bst LF (blue curves) or Bst 2.0 (black curves) on crude Covid-19 patient sample (prepared in QuickExtract). Amplification curves indicate real-time fluorescence measurements of RT-LAMP reactions (E1 primer set; in duplicates) using SARS-CoV-2 positive (filled circles) or SARS-CoV-2 negative (open circles) patient samples as input. B) Comparison of the ability of wildtype ( Bst LF, blue) and engineered Bst polymerase ( Bst 2.0, black) to incorporate dUTP during RT-LAMP on synthetic SARS-CoV-2 RNA standard. Reactions were either run under standard RT-LAMP conditions (-dUTP, filled circles), or supplemented with 0.7 mM dUTP, 0.7 mM dTTP and 1.4 mM of each dATP, dCTP, dGTP (open circles). Plotted is the ‘time to threshold’ as a measure of performance. C) LAMP performance (given as time to threshold in minutes) of indicated Bst DNA polymerase variants in the absence of a dedicated reverse transcriptase (-RTx) using diluted synthetic SARS-CoV-2 RNA (copies per reaction indicated) as template (related to Figure 6A ). D) RT-LAMP real-time fluorescence measurements using RTx and Bst 2.0 in IsoAmp buffer I (left) versus Bst 3.0 alone in IsoAmp buffer II (right). N2 DETECTR was used as primer set for amplifying synthetic SARS-CoV-2 RNA standard (copy number per reaction is indicated; no target control (NTC): water). E) Shown is the collateral cleavage activity (measured as real-time fluorescent signal) by Cas12, with a crRNA targeting the N2 LAMP amplicon, upon addition of 2 µ l of LAMP reactions from D) to 20 µ l of Cas12 cleavage mix. F) (Left) End-point fluorescence values (after 60 minutes) of RT-LAMP reactions from D). (Right) Cas12-based detection of LAMP products from D) is indicated.
    Figure Legend Snippet: Comparison of different Bst polymerases for RT-LAMP. A) Performance of Bst LF (blue curves) or Bst 2.0 (black curves) on crude Covid-19 patient sample (prepared in QuickExtract). Amplification curves indicate real-time fluorescence measurements of RT-LAMP reactions (E1 primer set; in duplicates) using SARS-CoV-2 positive (filled circles) or SARS-CoV-2 negative (open circles) patient samples as input. B) Comparison of the ability of wildtype ( Bst LF, blue) and engineered Bst polymerase ( Bst 2.0, black) to incorporate dUTP during RT-LAMP on synthetic SARS-CoV-2 RNA standard. Reactions were either run under standard RT-LAMP conditions (-dUTP, filled circles), or supplemented with 0.7 mM dUTP, 0.7 mM dTTP and 1.4 mM of each dATP, dCTP, dGTP (open circles). Plotted is the ‘time to threshold’ as a measure of performance. C) LAMP performance (given as time to threshold in minutes) of indicated Bst DNA polymerase variants in the absence of a dedicated reverse transcriptase (-RTx) using diluted synthetic SARS-CoV-2 RNA (copies per reaction indicated) as template (related to Figure 6A ). D) RT-LAMP real-time fluorescence measurements using RTx and Bst 2.0 in IsoAmp buffer I (left) versus Bst 3.0 alone in IsoAmp buffer II (right). N2 DETECTR was used as primer set for amplifying synthetic SARS-CoV-2 RNA standard (copy number per reaction is indicated; no target control (NTC): water). E) Shown is the collateral cleavage activity (measured as real-time fluorescent signal) by Cas12, with a crRNA targeting the N2 LAMP amplicon, upon addition of 2 µ l of LAMP reactions from D) to 20 µ l of Cas12 cleavage mix. F) (Left) End-point fluorescence values (after 60 minutes) of RT-LAMP reactions from D). (Right) Cas12-based detection of LAMP products from D) is indicated.

    Techniques Used: Amplification, Fluorescence, Activity Assay

    7) Product Images from "Ebolavirus polymerase uses an unconventional genome replication mechanism"

    Article Title: Ebolavirus polymerase uses an unconventional genome replication mechanism

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1815745116

    Mapping the 5′ end of EBOV RNAs by primer extension and sequence analysis. ( A and B ) Primer extension analysis of antigenome ( A ) and genome ( B ) RNAs. ( A and B , Upper ) Schematic diagram (not to scale) of the EBOV RNA that was analyzed showing the hybridization positions of the negative sense Le 14–35 ( A ) and positive sense Tr 17–41 ( B ) primers used for primer extension analysis. ( A and B , Lower ) Primer extension analysis. In each case, i shows analysis of RNA isolated from Vero cells infected with EBOV Mayinga (EBOV M ) or Kikwit (EBOV K ), and ii shows analysis of RNA isolated from EBOV Kikwit-infected Vero (nonhuman primate, NHP), Huh7 (human), and R05T (bat) cells. In each panel, [γ- 32 P]ATP end-labeled DNA oligonucleotides corresponding in length and sequence to cDNA representing initiation from positions +1 and +2, relative to the published EBOV sequences, were used as markers (lanes 1 and 2). ( C – H ) Vero cells were infected with EBOV Mayinga ( C – E ) or Kikwit ( F – H ) and total cellular or virion-associated RNA was used for RACE analysis. The traces show sequences of the 5′ RACE PCR population obtained from the 5′ ends of intracellular antigenomic viral RNA ( C and F ), intracellular genomic viral RNA ( D and G ), or virion-associated genomic RNA ( E and H ). The cDNA was tailed with dATP ( i ) or dCTP ( ii ). The black line below the sequence traces indicates poly(A) or poly(C) tail sequences that had been added to the virus-specific sequence during RACE. This may include some virus-specific sequence that cannot be distinguished from the poly(A) or poly(C) tail. The first two C residues that belong to the viral sequence are underlined with a dotted line in the poly(C)-tailed sequence traces.
    Figure Legend Snippet: Mapping the 5′ end of EBOV RNAs by primer extension and sequence analysis. ( A and B ) Primer extension analysis of antigenome ( A ) and genome ( B ) RNAs. ( A and B , Upper ) Schematic diagram (not to scale) of the EBOV RNA that was analyzed showing the hybridization positions of the negative sense Le 14–35 ( A ) and positive sense Tr 17–41 ( B ) primers used for primer extension analysis. ( A and B , Lower ) Primer extension analysis. In each case, i shows analysis of RNA isolated from Vero cells infected with EBOV Mayinga (EBOV M ) or Kikwit (EBOV K ), and ii shows analysis of RNA isolated from EBOV Kikwit-infected Vero (nonhuman primate, NHP), Huh7 (human), and R05T (bat) cells. In each panel, [γ- 32 P]ATP end-labeled DNA oligonucleotides corresponding in length and sequence to cDNA representing initiation from positions +1 and +2, relative to the published EBOV sequences, were used as markers (lanes 1 and 2). ( C – H ) Vero cells were infected with EBOV Mayinga ( C – E ) or Kikwit ( F – H ) and total cellular or virion-associated RNA was used for RACE analysis. The traces show sequences of the 5′ RACE PCR population obtained from the 5′ ends of intracellular antigenomic viral RNA ( C and F ), intracellular genomic viral RNA ( D and G ), or virion-associated genomic RNA ( E and H ). The cDNA was tailed with dATP ( i ) or dCTP ( ii ). The black line below the sequence traces indicates poly(A) or poly(C) tail sequences that had been added to the virus-specific sequence during RACE. This may include some virus-specific sequence that cannot be distinguished from the poly(A) or poly(C) tail. The first two C residues that belong to the viral sequence are underlined with a dotted line in the poly(C)-tailed sequence traces.

    Techniques Used: Sequencing, Hybridization, Isolation, Infection, Labeling, Polymerase Chain Reaction

    8) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.
    Figure Legend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Techniques Used: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    9) Product Images from "Visualizing Sequence-Governed Nucleotide Selectivities and Mutagenic Consequences through a Replicative Cycle: Processing of a Bulky Carcinogen N2-dG Lesion in a Y-family DNA olymerase †"

    Article Title: Visualizing Sequence-Governed Nucleotide Selectivities and Mutagenic Consequences through a Replicative Cycle: Processing of a Bulky Carcinogen N2-dG Lesion in a Y-family DNA olymerase †

    Journal: Biochemistry

    doi: 10.1021/bi802363f

    ; in addition, dATP/dA are navy blue, dGTP/dG are blue, and dCTP/dC are green. Views are after
    Figure Legend Snippet: ; in addition, dATP/dA are navy blue, dGTP/dG are blue, and dCTP/dC are green. Views are after

    Techniques Used:

    (A) Single dNTP insertion opposite G* (-1 → 0 step) catalyzed by Dpo4. Incorporation of each individual dNTP opposite G*, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and N is a mixture of all four nucleotides (4dNTPs). The dNTP concentrations
    Figure Legend Snippet: (A) Single dNTP insertion opposite G* (-1 → 0 step) catalyzed by Dpo4. Incorporation of each individual dNTP opposite G*, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and N is a mixture of all four nucleotides (4dNTPs). The dNTP concentrations

    Techniques Used:

    10) Product Images from "OTTER, a new method quantifying absolute amounts of tRNAs"

    Article Title: OTTER, a new method quantifying absolute amounts of tRNAs

    Journal: bioRxiv

    doi: 10.1101/2020.05.18.101501

    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    Figure Legend Snippet: tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

    Techniques Used: Fluorescence, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Northern Blot

    11) Product Images from "Using RecA Protein to Enhance Kinetic Rates of DNA Circuits †"

    Article Title: Using RecA Protein to Enhance Kinetic Rates of DNA Circuits †

    Journal: Chemical communications (Cambridge, England)

    doi: 10.1039/c5cc02261d

    Normalized kinetic rates for pre-annealed RecA with CHA. RecA protein was incubated with 10 nM Catalyst (C30) at 37°C for 10 min, then diluted down to a final concentration of 2.5 nM in the CHA reaction. The row marked ‘dATP’ indicates
    Figure Legend Snippet: Normalized kinetic rates for pre-annealed RecA with CHA. RecA protein was incubated with 10 nM Catalyst (C30) at 37°C for 10 min, then diluted down to a final concentration of 2.5 nM in the CHA reaction. The row marked ‘dATP’ indicates

    Techniques Used: Incubation, Concentration Assay

    Related Articles

    Purification:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Linearization of LIC expression vectors for LIC cloning LIC expression vectors (10 µg) were cut with 10 U PmeI in a 20-µL reaction volume and purified from contaminating stuffer fragment and undigested vector by gel-extraction using the NucleoSpin Extract II kit (Macherey & Nagel, Düren, Germany). .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and elution with 20 µL elution buffer included in the kit.

    Article Title: Ebolavirus polymerase uses an unconventional genome replication mechanism
    Article Snippet: .. Following purification, the cDNA was tailed with dCTP or dATP using terminal transferase (NEB). .. The tailed cDNA was PCR amplified using a nested virus-specific primer and a primer that annealed specifically with the dATP or the dCTP tail.

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Open reading frames of two cell wall degrading enzymes endo-β-1,4-glucanase (GenBank ID DQ490472) and endo-β-1,4-xylanase (DQ490490) were PCR amplified using genomic DNA from Pichia pastoris strains obtained from the Fungal Genetic Stock Centre (FGSC) . .. PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and eluting with 20 µL elution buffer included in the kit.

    Plasmid Preparation:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Linearization of LIC expression vectors for LIC cloning LIC expression vectors (10 µg) were cut with 10 U PmeI in a 20-µL reaction volume and purified from contaminating stuffer fragment and undigested vector by gel-extraction using the NucleoSpin Extract II kit (Macherey & Nagel, Düren, Germany). .. To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and elution with 20 µL elution buffer included in the kit.

    Amplification:

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing
    Article Snippet: Cells lysed in the home-made buffer (19.2 mM Tris–HCl (pH 7.8), 1 mM MgCl2 , 0.88 mM CaCl2, 20 μM DTT, 2% (wt/vol) Triton X-100) were incubated for 5 min at room temperature before incubation at 95°C for 5 min. For extracted RNA, RNA was purified from 1e5 HEK293 cells using standard Trizol RNA extraction and diluted to cell/reaction equivalents. .. dUTP/UDG contamination prevention system Reactions were set up to contain NEB 1x Isothermal Amplification Buffer, 1.4 mM of each dATP, dCTP, dGTP, 0.7 mM dUTP, 0.7 mM dTTP, 6 mM MgSO4 (100 mM stock, NEB), 0.32 U/µ l NEB Bst 2.0 polymerase, 0.3 U/µ l NEB Warmstart RTx Reverse Transcriptase, 0.2 U/µ l NEB Antarctic thermolabile UDG, sample and nuclease-free water. .. Reactions were set up on ice and incubated at room temperature for 5 minutes before being transferred to 63°C to start RT-LAMP reactions under standard conditions described above.

    other:

    Article Title: Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase
    Article Snippet: Dpn I Restriction enzyme, polynucleotide kinase (PNK), bovine serum albumin (BSA) and 100 mM stock solutions of dATP, dTTP, dGTP and dCTP were purchased from New England Biolabs.

    Polymerase Chain Reaction:

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection
    Article Snippet: Open reading frames of two cell wall degrading enzymes endo-β-1,4-glucanase (GenBank ID DQ490472) and endo-β-1,4-xylanase (DQ490490) were PCR amplified using genomic DNA from Pichia pastoris strains obtained from the Fungal Genetic Stock Centre (FGSC) . .. PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and eluting with 20 µL elution buffer included in the kit.

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    New England Biolabs datp
    Specificity of M. jannaschii PSTK for the phosphoryl donor. Initial velocities for the phosphotransferase activity of PSTK (6 nM PSTK without <t>NTP</t> or the addition of ATP, ITP, GTP, CTP, UTP or <t>dATP</t> or 200 nM PSTK with AMP-CPP or AMP-PCP) in the presence of the indicated phosphoryl donors (20 mM). Reactions proceeded for 6 min at 37°C and 1.5 min time points were taken and analyzed as in Figure 1 A and 2 . *ND, no detectable phosphorylation. Error bars represent the standard deviation of three experiments.
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    Specificity of M. jannaschii PSTK for the phosphoryl donor. Initial velocities for the phosphotransferase activity of PSTK (6 nM PSTK without NTP or the addition of ATP, ITP, GTP, CTP, UTP or dATP or 200 nM PSTK with AMP-CPP or AMP-PCP) in the presence of the indicated phosphoryl donors (20 mM). Reactions proceeded for 6 min at 37°C and 1.5 min time points were taken and analyzed as in Figure 1 A and 2 . *ND, no detectable phosphorylation. Error bars represent the standard deviation of three experiments.

    Journal: Nucleic Acids Research

    Article Title: Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

    doi: 10.1093/nar/gkm1134

    Figure Lengend Snippet: Specificity of M. jannaschii PSTK for the phosphoryl donor. Initial velocities for the phosphotransferase activity of PSTK (6 nM PSTK without NTP or the addition of ATP, ITP, GTP, CTP, UTP or dATP or 200 nM PSTK with AMP-CPP or AMP-PCP) in the presence of the indicated phosphoryl donors (20 mM). Reactions proceeded for 6 min at 37°C and 1.5 min time points were taken and analyzed as in Figure 1 A and 2 . *ND, no detectable phosphorylation. Error bars represent the standard deviation of three experiments.

    Article Snippet: For phosphoryl donor preference determination, the phosphotransferase assays were carried out for 6 min with 6 nM PSTK and either no NTP or 20 mM ultrapure ( > 99%) NTP (ATP, ITP, GTP, CTP, UTP) (Sigma) or dATP (New England Biolabs).

    Techniques: Activity Assay, Conditioned Place Preference, Standard Deviation

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Article Snippet: To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Techniques: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Article Snippet: To generate 5′ LIC overhangs (15 and 16 nt, respectively) at both ends the purified vector backbone was treated for 30 min (22°C) with T4 DNA polymerase in the presence of dATP, using the following reaction setup: 0.2 pmol purified vector backbone, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL dithiothreitol (DTT, 100 mM), 2 µL 10× (10 mg/mL) bovine serum albumin (BSA; NEB), 10 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Techniques: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    Simulated systems after 2 ns MD. Color code: light-pink, protein (ribbon representation); magenta, Tyr-714; grey, template DNA strand; cyan, primer DNA strand; orange, Mg 2+ ions (sphere representation); yellow, modified dG*; red, BP moiety; blue, dATP/dA; green, dCTP/dC; light blue, dGTP/dG; chocolate, dTTP/dT. ( k–m ) are the three simulated systems with [BP]G* at the post-insertion site of open BF: two computer-generated models ( k,1 ) and one crystal structure ( m ). The arrows in ( s ) indicate the motion of the Watson-Crick edges of modified guanine and partner C. The arrow in ( v ) indicates the movement of the phosphate of [BP]G*. Figure 4 is continued on the next page.

    Journal: Nucleic Acids Research

    Article Title: Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

    doi: 10.1093/nar/gkm416

    Figure Lengend Snippet: Simulated systems after 2 ns MD. Color code: light-pink, protein (ribbon representation); magenta, Tyr-714; grey, template DNA strand; cyan, primer DNA strand; orange, Mg 2+ ions (sphere representation); yellow, modified dG*; red, BP moiety; blue, dATP/dA; green, dCTP/dC; light blue, dGTP/dG; chocolate, dTTP/dT. ( k–m ) are the three simulated systems with [BP]G* at the post-insertion site of open BF: two computer-generated models ( k,1 ) and one crystal structure ( m ). The arrows in ( s ) indicate the motion of the Watson-Crick edges of modified guanine and partner C. The arrow in ( v ) indicates the movement of the phosphate of [BP]G*. Figure 4 is continued on the next page.

    Article Snippet: The dNTPs, dATP, dCTP, dGTP and dTTP were purchased from New England Biolabs, Inc. (Beverly, MA).

    Techniques: Modification, Generated, Polyacrylamide Gel Electrophoresis

    ( a ) Template-primer strand duplex used in the running start experiment showing the numbering scheme. ( b ) Typical running start primer extension experiments catalyzed by BF as a function of incubation time (min). Extension of the primer strand, a 22-mer with the terminal 3′-G base opposite the template base C labeled ‘–3’ on the template strand. The position of the [BP]G* is labeled ‘0’ (25-mer). The ‘–1’ position denotes a 24-mer. The lane marked ‘M’ contains 22-mer, 24-mer, 25-mer and 43-mer primer strands (the 43-mer corresponds to the length of a fully extended primer strand). Reactions were initiated with 4.5 nM of the primer/template duplex (1:1.5 ratio), 20 nM of BF, and 200 μM of dNTPs at 37°C. ( c ) Typical single step 2′-deoxynucleotide triphosphate (dNTP) insertion catalyzed by BF. The primer strand was 24 nucleotides long with the terminal 3′-C positioned opposite the template base labeled ‘–1’ which corresponds to the darkest bands in this figure. Incorporation of each individual dNTP opposite the [BP]G* adduct results in the lighter bands just above the darkest bands, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and Ctr = ‘–1’ and ‘0’ position marker. Reactions were initiated at 2 mM of each dNTP, 15 nM of [DNA], and 2 nM of BF at 37°C for 30 min.

    Journal: Nucleic Acids Research

    Article Title: Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

    doi: 10.1093/nar/gkm416

    Figure Lengend Snippet: ( a ) Template-primer strand duplex used in the running start experiment showing the numbering scheme. ( b ) Typical running start primer extension experiments catalyzed by BF as a function of incubation time (min). Extension of the primer strand, a 22-mer with the terminal 3′-G base opposite the template base C labeled ‘–3’ on the template strand. The position of the [BP]G* is labeled ‘0’ (25-mer). The ‘–1’ position denotes a 24-mer. The lane marked ‘M’ contains 22-mer, 24-mer, 25-mer and 43-mer primer strands (the 43-mer corresponds to the length of a fully extended primer strand). Reactions were initiated with 4.5 nM of the primer/template duplex (1:1.5 ratio), 20 nM of BF, and 200 μM of dNTPs at 37°C. ( c ) Typical single step 2′-deoxynucleotide triphosphate (dNTP) insertion catalyzed by BF. The primer strand was 24 nucleotides long with the terminal 3′-C positioned opposite the template base labeled ‘–1’ which corresponds to the darkest bands in this figure. Incorporation of each individual dNTP opposite the [BP]G* adduct results in the lighter bands just above the darkest bands, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and Ctr = ‘–1’ and ‘0’ position marker. Reactions were initiated at 2 mM of each dNTP, 15 nM of [DNA], and 2 nM of BF at 37°C for 30 min.

    Article Snippet: The dNTPs, dATP, dCTP, dGTP and dTTP were purchased from New England Biolabs, Inc. (Beverly, MA).

    Techniques: Incubation, Labeling, Marker

    The stalled leading strand is extended directly by TLS. A , pulse-chase, EcoRI runoff replication reactions were performed either in the absence or presence of elevated concentrations of dATP and dCTP using the CPD template as described under “Experimental procedures.” In these reactions, replication is initiated in the absence of DNA gyrase and allowed to proceed for 1 min when the replication forks stall because of the accumulation of positive supercoils. The template is then digested with EcoRI in less than 10 s at the same time as [α- 32 P]dGTP is added. After 1 min, the radiolabel is chased with a 100-fold excess of cold nucleotide. Time points are as indicated postchase. The products were analyzed by electrophoresis through either denaturing alkaline agarose gels ( top ) or native agarose gels ( bottom ). B , lane traces of the 1-, 3-, and 6-min lanes in the presence of elevated dATP and dCTP in A (denaturing). FL , full length; PSL , photostimulated luminescence.

    Journal: The Journal of Biological Chemistry

    Article Title: Replisome-mediated translesion synthesis by a cellular replicase

    doi: 10.1074/jbc.M117.800441

    Figure Lengend Snippet: The stalled leading strand is extended directly by TLS. A , pulse-chase, EcoRI runoff replication reactions were performed either in the absence or presence of elevated concentrations of dATP and dCTP using the CPD template as described under “Experimental procedures.” In these reactions, replication is initiated in the absence of DNA gyrase and allowed to proceed for 1 min when the replication forks stall because of the accumulation of positive supercoils. The template is then digested with EcoRI in less than 10 s at the same time as [α- 32 P]dGTP is added. After 1 min, the radiolabel is chased with a 100-fold excess of cold nucleotide. Time points are as indicated postchase. The products were analyzed by electrophoresis through either denaturing alkaline agarose gels ( top ) or native agarose gels ( bottom ). B , lane traces of the 1-, 3-, and 6-min lanes in the presence of elevated dATP and dCTP in A (denaturing). FL , full length; PSL , photostimulated luminescence.

    Article Snippet: Replication reactions (30 μl) were carried out as described above in the presence of an additional 750 μ m dATP and dCTP, and products were digested with EcoRI, PvuI, and DpnI (40 units) (New England Biolabs).

    Techniques: Pulse Chase, Electrophoresis

    Lesion bypass is directly proportional to nucleotide concentration. A , standard replication reactions with the indicated total concentrations of dATP ( dA ) and dCTP ( dC ) were analyzed by denaturing agarose gel electrophoresis. B , quantification of the results shown in A . Shown is the mean from two experiments. FL , full length.

    Journal: The Journal of Biological Chemistry

    Article Title: Replisome-mediated translesion synthesis by a cellular replicase

    doi: 10.1074/jbc.M117.800441

    Figure Lengend Snippet: Lesion bypass is directly proportional to nucleotide concentration. A , standard replication reactions with the indicated total concentrations of dATP ( dA ) and dCTP ( dC ) were analyzed by denaturing agarose gel electrophoresis. B , quantification of the results shown in A . Shown is the mean from two experiments. FL , full length.

    Article Snippet: Replication reactions (30 μl) were carried out as described above in the presence of an additional 750 μ m dATP and dCTP, and products were digested with EcoRI, PvuI, and DpnI (40 units) (New England Biolabs).

    Techniques: Concentration Assay, Agarose Gel Electrophoresis