Structured Review

Promega klenow dna polymerase
Klenow Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 11 article reviews
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klenow dna polymerase - by Bioz Stars, 2020-07
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Clone Assay:

Article Title: Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.
Article Snippet: .. Alternatively, clones were generated from cDNA synthesized using GAV-specific primers followed by second-strand synthesis randomly primed with UNIprimer-dN ' and extended using Klenow DNA polymerase (Froussard, 1992). cDNAs of variable length were amplified by PCR (Froussard, 1992) using both the virus-specific primer and UNI-primer and Taq DNA polymerase and buffer (Promega) containing 2±5 mM MgCl # . .. RT–PCR products were either gel purified or processed directly using QIAquick columns and cloned into either pGEM-T vector or EcoRV-cut pBluescript II KS().

Amplification:

Article Title: Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.
Article Snippet: .. Alternatively, clones were generated from cDNA synthesized using GAV-specific primers followed by second-strand synthesis randomly primed with UNIprimer-dN ' and extended using Klenow DNA polymerase (Froussard, 1992). cDNAs of variable length were amplified by PCR (Froussard, 1992) using both the virus-specific primer and UNI-primer and Taq DNA polymerase and buffer (Promega) containing 2±5 mM MgCl # . .. RT–PCR products were either gel purified or processed directly using QIAquick columns and cloned into either pGEM-T vector or EcoRV-cut pBluescript II KS().

Synthesized:

Article Title: Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.
Article Snippet: .. Alternatively, clones were generated from cDNA synthesized using GAV-specific primers followed by second-strand synthesis randomly primed with UNIprimer-dN ' and extended using Klenow DNA polymerase (Froussard, 1992). cDNAs of variable length were amplified by PCR (Froussard, 1992) using both the virus-specific primer and UNI-primer and Taq DNA polymerase and buffer (Promega) containing 2±5 mM MgCl # . .. RT–PCR products were either gel purified or processed directly using QIAquick columns and cloned into either pGEM-T vector or EcoRV-cut pBluescript II KS().

Article Title: Non-IG Aberrations of FOXP1 in B-Cell Malignancies Lead to an Aberrant Expression of N-Truncated Isoforms of FOXP1
Article Snippet: .. Second strand was synthesized using Klenow DNA polymerase (Promega, Fitchburg, WI, USA) and the primers mix TV8. .. Anchored PCR was performed for 35 cycles with primers FOXP1/ex7R/race-1 and 467, and for nested PCR we used the primers FOXP1/ex7R/race-2 and 468.

Generated:

Article Title: Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.
Article Snippet: .. Alternatively, clones were generated from cDNA synthesized using GAV-specific primers followed by second-strand synthesis randomly primed with UNIprimer-dN ' and extended using Klenow DNA polymerase (Froussard, 1992). cDNAs of variable length were amplified by PCR (Froussard, 1992) using both the virus-specific primer and UNI-primer and Taq DNA polymerase and buffer (Promega) containing 2±5 mM MgCl # . .. RT–PCR products were either gel purified or processed directly using QIAquick columns and cloned into either pGEM-T vector or EcoRV-cut pBluescript II KS().

Labeling:

Article Title: Trans-spliced leader addition to mRNAs in a cnidarian
Article Snippet: .. Probes labeled with [α-32 P]dATP were made by extending a primer annealed to a synthetic oligonucleotide template by using Klenow DNA polymerase (Promega) in Prime-It II dATP reaction buffer (Stratagene). ..

Article Title: Cell confluency-induced Stat3 activation regulates NHE3 expression by recruiting Sp1 and Sp3 to the proximal NHE3 promoter region during epithelial dome formation
Article Snippet: .. The Sp binding oligonucleotide WTABC: 5′-AGCGGG GGCG GGAAGGCCC CGCC CTGGCGCG GGAG GGGGCAG-3′ and the Stat3-specific binding oligonucleotide M67: 5′-TAGGGAT TTACGGGAA ATGAAGCT-3′ were labeled with [α-32 P]dCTP by the filled-in reaction with Klenow DNA polymerase (Promega, Madison, WI). ..

Article Title: The LysR-Type Transcriptional Regulator VirR Is Required for Expression of the Virulence Gene vapA of Rhodococcus equi ATCC 33701
Article Snippet: .. To obtain a radiolabeled DNA fragment containing the vapA promoter region, we digested pEDAR1012 with BglII and HindIII and labeled the resulting 262-bp DNA fragment with [α-32 P]dATP (Perkin-Elmer) in a mixture containing 50 ng of DNA, 100 μM dCTP, 100 μM dGTP, 100 μM dTTP, 5 μCi of [α-32 P]dATP, 2 U of Klenow DNA polymerase (Promega), 50 mM Tris-HCl (pH 7.2), 10 mM MgSO4 , and 0.1 mM DTT which was then incubated at 30°C for 30 min. ..

Purification:

Article Title: Interaction of Ler at the LEE5 (tir) Operon of Enteropathogenic Escherichia coli
Article Snippet: .. The purified plasmid DNA was cut with Not I restriction endonuclease, which left a 5′ overhang that was subsequently filled in by using [α-32 P]dCTP (Amersham), dGTP, and Klenow DNA polymerase (Promega). .. The plasmid DNA was then cut with Eco RI, releasing the LEE5 regulatory fragments.

Incubation:

Article Title: The LysR-Type Transcriptional Regulator VirR Is Required for Expression of the Virulence Gene vapA of Rhodococcus equi ATCC 33701
Article Snippet: .. To obtain a radiolabeled DNA fragment containing the vapA promoter region, we digested pEDAR1012 with BglII and HindIII and labeled the resulting 262-bp DNA fragment with [α-32 P]dATP (Perkin-Elmer) in a mixture containing 50 ng of DNA, 100 μM dCTP, 100 μM dGTP, 100 μM dTTP, 5 μCi of [α-32 P]dATP, 2 U of Klenow DNA polymerase (Promega), 50 mM Tris-HCl (pH 7.2), 10 mM MgSO4 , and 0.1 mM DTT which was then incubated at 30°C for 30 min. ..

Polymerase Chain Reaction:

Article Title: Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.
Article Snippet: .. Alternatively, clones were generated from cDNA synthesized using GAV-specific primers followed by second-strand synthesis randomly primed with UNIprimer-dN ' and extended using Klenow DNA polymerase (Froussard, 1992). cDNAs of variable length were amplified by PCR (Froussard, 1992) using both the virus-specific primer and UNI-primer and Taq DNA polymerase and buffer (Promega) containing 2±5 mM MgCl # . .. RT–PCR products were either gel purified or processed directly using QIAquick columns and cloned into either pGEM-T vector or EcoRV-cut pBluescript II KS().

Binding Assay:

Article Title: Cell confluency-induced Stat3 activation regulates NHE3 expression by recruiting Sp1 and Sp3 to the proximal NHE3 promoter region during epithelial dome formation
Article Snippet: .. The Sp binding oligonucleotide WTABC: 5′-AGCGGG GGCG GGAAGGCCC CGCC CTGGCGCG GGAG GGGGCAG-3′ and the Stat3-specific binding oligonucleotide M67: 5′-TAGGGAT TTACGGGAA ATGAAGCT-3′ were labeled with [α-32 P]dCTP by the filled-in reaction with Klenow DNA polymerase (Promega, Madison, WI). ..

Plasmid Preparation:

Article Title: Interaction of Ler at the LEE5 (tir) Operon of Enteropathogenic Escherichia coli
Article Snippet: .. The purified plasmid DNA was cut with Not I restriction endonuclease, which left a 5′ overhang that was subsequently filled in by using [α-32 P]dCTP (Amersham), dGTP, and Klenow DNA polymerase (Promega). .. The plasmid DNA was then cut with Eco RI, releasing the LEE5 regulatory fragments.

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  • 85
    Promega e coli exo free pol i
    Pol θ extends from an A opposite the second T of a (6–4) photoproduct (A) A 17-mer (left, lanes 1–12) or an 18-mer (right, lanes 13–24) of the DNA sequence shown was labeled with 32 P at the 5’ end (denoted by the asterisk) and annealed to template 30-mer DNA containing either a (6–4)PP at a TT sequence, or normal TT bases at the same sequence (undamaged). Extension from an A opposite a T of an undamaged template (lanes 2–6) or the first T of a (6–4)PP (lanes 8–12); extension from an A opposite the T of an undamaged template (lanes 14–18) or the second T of a (6–4)PP (lanes 20–24). A time course assay was performed and control experiments without pol θ are shown in lanes 1, 7, 13, and 19. (B) <t>exo-free</t> Klenow fragment of E. coli <t>pol</t> I does not extend from an A opposite a (6–4)PP or a CPD. Extension was tested with 9 x 10 −3 Unit (x 1) and 9 x 10 −2 Unit (x 10) of enzyme at 37°C for 10 min. Extension reactions from an A opposite the first T of a CPD (lanes 2 and 3), opposite the first T of a (6–4)PP (lanes 5 and 6) and opposite the second T of a (6–4)PP (lanes 8 and 9) were examined. Reaction mixtures with no enzyme are shown in lanes 1, 4 and 7.
    E Coli Exo Free Pol I, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli exo free pol i/product/Promega
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli exo free pol i - by Bioz Stars, 2020-07
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    86
    Promega klenow dna polymerase enzymes
    Differences of topoisomerase I plasmid cleavage activity trapped by TpT in liver samples and in hepatoma cells (representative image of three independent experiments). pBR322 plasmid was linearized and end-labeled with 5 μ Ci α 32 P ATP by using 20 U <t>Klenow</t> <t>DNA</t> polymerase. To remove the labeling from one end, the linearized plasmid was digested with Hind III restriction endonuclease. Ten microgram 0.35 M NaCI nuclear extracts of HepG2, Hep3B, and human HCC specimens were incubated with 8000 cpm linearized plasmid in the absence or presence of 100 and 200 μ M topotecan. Lanes 1: cleavage reaction without topotecan, lane 2: cleavage reaction with 100 μ M topotecan, and lane 3: cleavage reaction with 200 μ M topotecan. The activity of Hep3B extract was much lower than that of HepG2 and human Iiver cancer.
    Klenow Dna Polymerase Enzymes, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow dna polymerase enzymes/product/Promega
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    klenow dna polymerase enzymes - by Bioz Stars, 2020-07
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    93
    Promega klenow fragment
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 72 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Pol θ extends from an A opposite the second T of a (6–4) photoproduct (A) A 17-mer (left, lanes 1–12) or an 18-mer (right, lanes 13–24) of the DNA sequence shown was labeled with 32 P at the 5’ end (denoted by the asterisk) and annealed to template 30-mer DNA containing either a (6–4)PP at a TT sequence, or normal TT bases at the same sequence (undamaged). Extension from an A opposite a T of an undamaged template (lanes 2–6) or the first T of a (6–4)PP (lanes 8–12); extension from an A opposite the T of an undamaged template (lanes 14–18) or the second T of a (6–4)PP (lanes 20–24). A time course assay was performed and control experiments without pol θ are shown in lanes 1, 7, 13, and 19. (B) exo-free Klenow fragment of E. coli pol I does not extend from an A opposite a (6–4)PP or a CPD. Extension was tested with 9 x 10 −3 Unit (x 1) and 9 x 10 −2 Unit (x 10) of enzyme at 37°C for 10 min. Extension reactions from an A opposite the first T of a CPD (lanes 2 and 3), opposite the first T of a (6–4)PP (lanes 5 and 6) and opposite the second T of a (6–4)PP (lanes 8 and 9) were examined. Reaction mixtures with no enzyme are shown in lanes 1, 4 and 7.

    Journal: DNA repair

    Article Title: DNA polymerase ? (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

    doi: 10.1016/j.dnarep.2007.08.005

    Figure Lengend Snippet: Pol θ extends from an A opposite the second T of a (6–4) photoproduct (A) A 17-mer (left, lanes 1–12) or an 18-mer (right, lanes 13–24) of the DNA sequence shown was labeled with 32 P at the 5’ end (denoted by the asterisk) and annealed to template 30-mer DNA containing either a (6–4)PP at a TT sequence, or normal TT bases at the same sequence (undamaged). Extension from an A opposite a T of an undamaged template (lanes 2–6) or the first T of a (6–4)PP (lanes 8–12); extension from an A opposite the T of an undamaged template (lanes 14–18) or the second T of a (6–4)PP (lanes 20–24). A time course assay was performed and control experiments without pol θ are shown in lanes 1, 7, 13, and 19. (B) exo-free Klenow fragment of E. coli pol I does not extend from an A opposite a (6–4)PP or a CPD. Extension was tested with 9 x 10 −3 Unit (x 1) and 9 x 10 −2 Unit (x 10) of enzyme at 37°C for 10 min. Extension reactions from an A opposite the first T of a CPD (lanes 2 and 3), opposite the first T of a (6–4)PP (lanes 5 and 6) and opposite the second T of a (6–4)PP (lanes 8 and 9) were examined. Reaction mixtures with no enzyme are shown in lanes 1, 4 and 7.

    Article Snippet: E. coli exo free pol I (Klenow fragment, Kf) was purchased from Promega Corporation, Madison, Wisconsin (catalog number M2181) and the supplied pol I buffer was used for the assays (50 mM Tris-HCl [pH 7.2], 10 mM MgSO4 , 0.1 mM dithiothreitol).

    Techniques: Sequencing, Labeling

    Differences of topoisomerase I plasmid cleavage activity trapped by TpT in liver samples and in hepatoma cells (representative image of three independent experiments). pBR322 plasmid was linearized and end-labeled with 5 μ Ci α 32 P ATP by using 20 U Klenow DNA polymerase. To remove the labeling from one end, the linearized plasmid was digested with Hind III restriction endonuclease. Ten microgram 0.35 M NaCI nuclear extracts of HepG2, Hep3B, and human HCC specimens were incubated with 8000 cpm linearized plasmid in the absence or presence of 100 and 200 μ M topotecan. Lanes 1: cleavage reaction without topotecan, lane 2: cleavage reaction with 100 μ M topotecan, and lane 3: cleavage reaction with 200 μ M topotecan. The activity of Hep3B extract was much lower than that of HepG2 and human Iiver cancer.

    Journal: BioMed Research International

    Article Title: Heparin and Liver Heparan Sulfate Can Rescue Hepatoma Cells from Topotecan Action

    doi: 10.1155/2014/765794

    Figure Lengend Snippet: Differences of topoisomerase I plasmid cleavage activity trapped by TpT in liver samples and in hepatoma cells (representative image of three independent experiments). pBR322 plasmid was linearized and end-labeled with 5 μ Ci α 32 P ATP by using 20 U Klenow DNA polymerase. To remove the labeling from one end, the linearized plasmid was digested with Hind III restriction endonuclease. Ten microgram 0.35 M NaCI nuclear extracts of HepG2, Hep3B, and human HCC specimens were incubated with 8000 cpm linearized plasmid in the absence or presence of 100 and 200 μ M topotecan. Lanes 1: cleavage reaction without topotecan, lane 2: cleavage reaction with 100 μ M topotecan, and lane 3: cleavage reaction with 200 μ M topotecan. The activity of Hep3B extract was much lower than that of HepG2 and human Iiver cancer.

    Article Snippet: Hind III and Klenow DNA polymerase enzymes were obtained from Promega (Madison, USA).

    Techniques: Plasmid Preparation, Activity Assay, Labeling, Incubation

    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration

    Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: End Labeling, Labeling

    DNA replication fidelity of DNA polymerase I. ( A ) dITP incorporation opposite dN residues of template DNA by DNA polymerase I. 5′-End labeling of 15mer primer DNA annealed to template DNA (top sequences) was used. dITP incorporation into the dN template was with 1 U DNA polymerase (exonuclease – ) and 100 µM dITP in 20 µl of reaction mixture at 37°C for 15 min. Reaction mixtures were separated by denaturing PAGE and autoradiograms were obtained using a BAS2000 image analyzer. ( B ) dNTP incorporation opposite HX residues of template DNA by DNA polymerase I. 5′-End-labeling of 16mer primer DNA annealed to template DNAs (top sequences) was used in this experiment. dNTP incorporation into the HX template was with 1 U Klenow fragment and 1–100 µM dNTP in 20 µl of reaction mixture at 37°C for 15 min. Lane C, control; lane 1, 1 µM dNTP; lane 2, 10 µM dNTP; lane 3, 100 µM dNTP.

    Journal: Nucleic Acids Research

    Article Title: Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii

    doi:

    Figure Lengend Snippet: DNA replication fidelity of DNA polymerase I. ( A ) dITP incorporation opposite dN residues of template DNA by DNA polymerase I. 5′-End labeling of 15mer primer DNA annealed to template DNA (top sequences) was used. dITP incorporation into the dN template was with 1 U DNA polymerase (exonuclease – ) and 100 µM dITP in 20 µl of reaction mixture at 37°C for 15 min. Reaction mixtures were separated by denaturing PAGE and autoradiograms were obtained using a BAS2000 image analyzer. ( B ) dNTP incorporation opposite HX residues of template DNA by DNA polymerase I. 5′-End-labeling of 16mer primer DNA annealed to template DNAs (top sequences) was used in this experiment. dNTP incorporation into the HX template was with 1 U Klenow fragment and 1–100 µM dNTP in 20 µl of reaction mixture at 37°C for 15 min. Lane C, control; lane 1, 1 µM dNTP; lane 2, 10 µM dNTP; lane 3, 100 µM dNTP.

    Article Snippet: We also observed that HX, when present in template DNA, directs incorporation of all four dNTPs by Klenow fragment with similar rates (Fig. B).

    Techniques: End Labeling, Polyacrylamide Gel Electrophoresis