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  • 99
    Kapa Biosystems kapa hifi dna polymerase
    RNA-Seq mapping statistics. Libraries were prepared either from polyA-enriched (polyA) or Tex-treated RNA (Tex). <t>DNA</t> was amplified using the DNA polymerases <t>KAPA</t> <t>Hifi</t> (Kapabiosystems) or Platinum® Pfx (PFX, Invitrogen).
    Kapa Hifi Dna Polymerase, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 1359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dntps
    Thermal denaturation transitions of duplexes 9 : 5 – 9 : 8 and H-bonding patterns expected from I, 8-oxoI, and 8-BrI (left); and RTn using <t>AMV</t> on duplexes 9 : 5 – 9 : 8 in the presence of canonical <t>dNTPs,</t> where M = equimolar mixture of all dNTPs, (right). Steady-state kinetics data is shown in the lower left corner.
    Dntps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dntp
    Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp <t>DNA</t> size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each <t>dNTP,</t> 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.
    Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 43505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dntp mix
    Vpx+ virus, dN and <t>dNTP</t> treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or <t>dNTPs,</t> and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.
    Dntp Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp  (TaKaRa)
    99
    TaKaRa dntp
    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), <t>10×</t> buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L <t>dNTP</t> (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.
    Dntp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 14874 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega dntps
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Dntps, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 18261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dntp mix
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Dntp Mix, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Meridian Life Science dntps
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Dntps, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 94/100, based on 3031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa dntp mix
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Dntp Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 3041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dntps
    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using <t>BrdUTP</t> alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without <t>dNTPs</t> and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.
    Dntps, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4077 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare dntps
    Pol V Mut is not activated by GTP, ADP, or dTTP and does not incorporate <t>ATP/ATPγS</t> in to DNA during synthesis. Pol V Mut WT, pol V Mut E38K/K72R, and pol V Mut E38K/ΔC17 were assembled according to the protocol in Figure 1A . ( A ) ATP, dATP, GTP, ADP, dTTP or ATPγS (500 μM) were used to activate pol V Mut for DNA synthesis and activity was checked in the presence of <t>dNTPs</t> (500 μM) and 3 nt oh HP (50 nM). ( B ) A HP containing TTT as its 3 nt oh was employed to determine if the various pol V Muts insert ATP or ATPγS during DNA synthesis. DNA extension is only observed in reactions where dATP is included. Other dNTPs are not present in any of the reactions. DOI: http://dx.doi.org/10.7554/eLife.02384.004
    Dntps, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2671 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Stratagene dntps
    Pol V Mut is not activated by GTP, ADP, or dTTP and does not incorporate <t>ATP/ATPγS</t> in to DNA during synthesis. Pol V Mut WT, pol V Mut E38K/K72R, and pol V Mut E38K/ΔC17 were assembled according to the protocol in Figure 1A . ( A ) ATP, dATP, GTP, ADP, dTTP or ATPγS (500 μM) were used to activate pol V Mut for DNA synthesis and activity was checked in the presence of <t>dNTPs</t> (500 μM) and 3 nt oh HP (50 nM). ( B ) A HP containing TTT as its 3 nt oh was employed to determine if the various pol V Muts insert ATP or ATPγS during DNA synthesis. DNA extension is only observed in reactions where dATP is included. Other dNTPs are not present in any of the reactions. DOI: http://dx.doi.org/10.7554/eLife.02384.004
    Dntps, supplied by Stratagene, used in various techniques. Bioz Stars score: 94/100, based on 1476 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega deoxynucleotide triphosphates
    Primer extension with <t>deoxynucleotide</t> triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.
    Deoxynucleotide Triphosphates, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Toyobo dntps
    Primer extension with <t>deoxynucleotide</t> triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.
    Dntps, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 1247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Kapa Biosystems kapa hifi hotstart dna polymerase
    Primer extension with <t>deoxynucleotide</t> triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.
    Kapa Hifi Hotstart Dna Polymerase, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 571 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad dntps
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntps, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RNA-Seq mapping statistics. Libraries were prepared either from polyA-enriched (polyA) or Tex-treated RNA (Tex). DNA was amplified using the DNA polymerases KAPA Hifi (Kapabiosystems) or Platinum® Pfx (PFX, Invitrogen).

    Journal: BMC Genomics

    Article Title: Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum

    doi: 10.1186/1471-2164-15-150

    Figure Lengend Snippet: RNA-Seq mapping statistics. Libraries were prepared either from polyA-enriched (polyA) or Tex-treated RNA (Tex). DNA was amplified using the DNA polymerases KAPA Hifi (Kapabiosystems) or Platinum® Pfx (PFX, Invitrogen).

    Article Snippet: 20 μM) was used instead of the RNA adapter provided by Illumina, the PCR amplification was performed for 12-16 cycles using the KAPA HiFi DNA polymerase (Kapabiosystems) and KAPA HiFi Fidelity Buffer according the manufacturer’s instructions (Mg2 concentration was adjusted to 2.5 mM).

    Techniques: RNA Sequencing Assay, Amplification

    Flowchart of strand-specific RNA-Seq library preparation. A) Strand-specific sequencing libraries are prepared from total RNA depleted of rRNA by digestion of 5′-P-containing RNA fragments with a 5′-phosphate-dependent exonuclease (Tex) or by enrichment for polyadenylated mRNA. Subsequently, rRNA-depleted RNA is decapped, fragmented, dephosphorylated and re-phosphorylated to obtain RNA fragments with 5′-P and 3′-OH groups. Next, the RNA is ligated to 3′ and 5′-adapters, reverse-transcribed with a primer complementary to the 3′-adapter and the cDNA is PCR amplified for 12-16 cycles using KAPA HiFi polymerase. A recent analysis had compared amplification efficiencies of different polymerases for genomes ranging in GC-content from 67.7% ( Boretella pertussis ) to 19.3% ( P. falciparum ]. B) Meta-gene coverage plots for libraries prepared from polyA-enriched and Tex-treated RNA. X-axis: scaled gene body, y-axis: scaled coverage.

    Journal: BMC Genomics

    Article Title: Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum

    doi: 10.1186/1471-2164-15-150

    Figure Lengend Snippet: Flowchart of strand-specific RNA-Seq library preparation. A) Strand-specific sequencing libraries are prepared from total RNA depleted of rRNA by digestion of 5′-P-containing RNA fragments with a 5′-phosphate-dependent exonuclease (Tex) or by enrichment for polyadenylated mRNA. Subsequently, rRNA-depleted RNA is decapped, fragmented, dephosphorylated and re-phosphorylated to obtain RNA fragments with 5′-P and 3′-OH groups. Next, the RNA is ligated to 3′ and 5′-adapters, reverse-transcribed with a primer complementary to the 3′-adapter and the cDNA is PCR amplified for 12-16 cycles using KAPA HiFi polymerase. A recent analysis had compared amplification efficiencies of different polymerases for genomes ranging in GC-content from 67.7% ( Boretella pertussis ) to 19.3% ( P. falciparum ]. B) Meta-gene coverage plots for libraries prepared from polyA-enriched and Tex-treated RNA. X-axis: scaled gene body, y-axis: scaled coverage.

    Article Snippet: 20 μM) was used instead of the RNA adapter provided by Illumina, the PCR amplification was performed for 12-16 cycles using the KAPA HiFi DNA polymerase (Kapabiosystems) and KAPA HiFi Fidelity Buffer according the manufacturer’s instructions (Mg2 concentration was adjusted to 2.5 mM).

    Techniques: RNA Sequencing Assay, Sequencing, Polymerase Chain Reaction, Amplification

    Thermal denaturation transitions of duplexes 9 : 5 – 9 : 8 and H-bonding patterns expected from I, 8-oxoI, and 8-BrI (left); and RTn using AMV on duplexes 9 : 5 – 9 : 8 in the presence of canonical dNTPs, where M = equimolar mixture of all dNTPs, (right). Steady-state kinetics data is shown in the lower left corner.

    Journal: bioRxiv

    Article Title: Translesion Synthesis by MmLV-, AMV-, and HIV-Reverse Transcriptases Using RNA Templates Containing Inosine, Guanosine, and Their 8-oxo-7,8-Dihydropurine Derivatives

    doi: 10.1101/2020.06.10.144048

    Figure Lengend Snippet: Thermal denaturation transitions of duplexes 9 : 5 – 9 : 8 and H-bonding patterns expected from I, 8-oxoI, and 8-BrI (left); and RTn using AMV on duplexes 9 : 5 – 9 : 8 in the presence of canonical dNTPs, where M = equimolar mixture of all dNTPs, (right). Steady-state kinetics data is shown in the lower left corner.

    Article Snippet: Reactions corresponding to the HIV-RT were carried out using the buffer for AMV-RT. dNTPs (dGTP, dCTP, dATP, dTTP) were purchased from Thermo Fisher or New England Biolabs (at a concentration of 100 mM) and diluted to a final concentration of 1.7 or 0.5 mM per experiment (single tube and well loaded onto the gel). dNTP mix, typically labeled M on subsequent figures, was diluted to a final concentration of 425 μM in each dNTP per experiment.

    Techniques:

    Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

    Journal: AoB Plants

    Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

    doi: 10.1093/aobpla/pls033

    Figure Lengend Snippet: Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

    Article Snippet: All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

    Techniques: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

    Journal: AoB Plants

    Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

    doi: 10.1093/aobpla/pls033

    Figure Lengend Snippet: Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

    Article Snippet: All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

    Techniques: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Vpx+ virus, dN and dNTP treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or dNTPs, and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.

    Journal: Stem cell research

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

    doi: 10.1016/j.scr.2015.06.012

    Figure Lengend Snippet: Vpx+ virus, dN and dNTP treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or dNTPs, and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.

    Article Snippet: THP-1 cells transduced with lentiviral vectors encoding SAMHD1 shRNA were selected by culturing in a medium containing 0.8 μg/ml puromycin. dNs consisted of a mixture of dA (D8668), dC (D0776), dG (D0901) and dT (T1895; all from Sigma-Aldrich). dNTPs (R0192) were purchased from Fermentas (Glen Burnie, MD).

    Techniques: Isolation, Cell Culture, Lysis, Two Tailed Test

    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells

    doi:

    Figure Lengend Snippet: Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Journal: Molecules

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    doi: 10.3390/molecules21060766

    Figure Lengend Snippet: Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Article Snippet: Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio).

    Techniques: Activity Assay, Labeling, Incubation

    Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Journal: Molecules

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    doi: 10.3390/molecules21060766

    Figure Lengend Snippet: Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Article Snippet: Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio).

    Techniques: Labeling, Incubation

    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Journal: BMC Biochemistry

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    doi: 10.1186/1471-2091-12-47

    Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Article Snippet: BrdUTP and dNTPs were from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

    Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Journal: Nucleic Acids Research

    Article Title: Mechanical properties of DNA-like polymers

    doi: 10.1093/nar/gkt808

    Figure Lengend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Article Snippet: For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

    Techniques: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

    Pol V Mut is not activated by GTP, ADP, or dTTP and does not incorporate ATP/ATPγS in to DNA during synthesis. Pol V Mut WT, pol V Mut E38K/K72R, and pol V Mut E38K/ΔC17 were assembled according to the protocol in Figure 1A . ( A ) ATP, dATP, GTP, ADP, dTTP or ATPγS (500 μM) were used to activate pol V Mut for DNA synthesis and activity was checked in the presence of dNTPs (500 μM) and 3 nt oh HP (50 nM). ( B ) A HP containing TTT as its 3 nt oh was employed to determine if the various pol V Muts insert ATP or ATPγS during DNA synthesis. DNA extension is only observed in reactions where dATP is included. Other dNTPs are not present in any of the reactions. DOI: http://dx.doi.org/10.7554/eLife.02384.004

    Journal: eLife

    Article Title: DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase

    doi: 10.7554/eLife.02384

    Figure Lengend Snippet: Pol V Mut is not activated by GTP, ADP, or dTTP and does not incorporate ATP/ATPγS in to DNA during synthesis. Pol V Mut WT, pol V Mut E38K/K72R, and pol V Mut E38K/ΔC17 were assembled according to the protocol in Figure 1A . ( A ) ATP, dATP, GTP, ADP, dTTP or ATPγS (500 μM) were used to activate pol V Mut for DNA synthesis and activity was checked in the presence of dNTPs (500 μM) and 3 nt oh HP (50 nM). ( B ) A HP containing TTT as its 3 nt oh was employed to determine if the various pol V Muts insert ATP or ATPγS during DNA synthesis. DNA extension is only observed in reactions where dATP is included. Other dNTPs are not present in any of the reactions. DOI: http://dx.doi.org/10.7554/eLife.02384.004

    Article Snippet: Pol V Mut (400 nM) was added to a 10-μl reaction mixture containing annealed template DNA (50 nM), ATP, ATPγS, dATP, GTP, dTTP, or ADP (500 μM, unless stated otherwise) and dNTPs (Amersham-Pharmacia) (500 μM each).

    Techniques: DNA Synthesis, Activity Assay

    Pol V Mut WT activity on DNA containing an abasic site. Pol V Mut (400 nM) activity was detected on 5′- 32 P-labeled 12 nt oh HP (100 nM), containing an abasic site 3 nts upstream from the 3′-OH, in the presence or absence of ATP/ATPγS and dNTPs. Lesion bypass is only observed when ATPγS is present. DOI: http://dx.doi.org/10.7554/eLife.02384.005

    Journal: eLife

    Article Title: DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent ATPase

    doi: 10.7554/eLife.02384

    Figure Lengend Snippet: Pol V Mut WT activity on DNA containing an abasic site. Pol V Mut (400 nM) activity was detected on 5′- 32 P-labeled 12 nt oh HP (100 nM), containing an abasic site 3 nts upstream from the 3′-OH, in the presence or absence of ATP/ATPγS and dNTPs. Lesion bypass is only observed when ATPγS is present. DOI: http://dx.doi.org/10.7554/eLife.02384.005

    Article Snippet: Pol V Mut (400 nM) was added to a 10-μl reaction mixture containing annealed template DNA (50 nM), ATP, ATPγS, dATP, GTP, dTTP, or ADP (500 μM, unless stated otherwise) and dNTPs (Amersham-Pharmacia) (500 μM each).

    Techniques: Activity Assay, Labeling

    Primer extension with deoxynucleotide triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.

    Journal: Nucleic Acids Research

    Article Title: Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays

    doi:

    Figure Lengend Snippet: Primer extension with deoxynucleotide triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.

    Article Snippet: Deoxynucleotide triphosphates (Promega), dideoxynucleotide triphosphates (MBI) and acyclonucleotide triphosphates (NEB) were purchased commercially.

    Techniques:

    Incorporation of Fc1-dUTP into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of dNTPs as indicated. DNA fragment lengths in nucleotides are shown on the left.

    Journal: Nucleic Acids Research

    Article Title: Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

    doi:

    Figure Lengend Snippet: Incorporation of Fc1-dUTP into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of dNTPs as indicated. DNA fragment lengths in nucleotides are shown on the left.

    Article Snippet: To generate the duplex for HPLC–ECD, the P18.1 and T40.1 oligonucleotides (4 µM each) were annealed before incubation in 240 µl of reaction mixture containing 6.7 mM Tris–HCl pH 8.8, 6.6 mM MgCl2 , 1 mM DTT, 16.8 mM (NH4 )2 SO4 , 200 µM dNTPs (except dTTP), 200 µM Fc1-dUTP and 0.25 U/µl Klenow fragment at room temperature for 20 min. Low molecular weight components were removed with a Bio-Spin 30 chromatography column (Bio-Rad).

    Techniques: Incubation