Structured Review

Promega klenow dna polymerase fragment
Klenow Dna Polymerase Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 88 stars, based on 2 article reviews
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klenow dna polymerase fragment - by Bioz Stars, 2020-07
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Plasmid Preparation:

Article Title: Syndecan-1 regulates ?v?3 and ?v?5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor
Article Snippet: .. The BstEII overhang was then filled in using the Klenow DNA polymerase fragment (Promega) and the vector then blunt-end ligated using T4 DNA ligase (New England Biolabs, Inc.). .. The mutant was transfected into MDA-MB-231 cells using LipofectAMINE PLUS (Invitrogen) and 10 µg of plasmid, in accordance with the manufacturer's instructions.

Article Title: Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation
Article Snippet: .. It was created by excising the CMV promoter region from the pCI vector (Promega) with Bgl II and Nhe I, blunting the ends with the Klenow DNA polymerase fragment and inserting the promoter fragment into Sma I-cleaved pGL3-Basic vector (Promega). ..

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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
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    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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    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
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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

    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

    Single-stranded nucleic acid ends are required to inhibit escape commitment. (A) Method used to monitor escape commitment. Nucleic acid inhibitors were added to transcription reaction mixtures at points 1, 2, and 3. (B) Poly(dI-dC) does not inhibit escape commitment. Poly(dI-dC) and ctDNA were added to transcription reactions at the points indicated (see panel A for the method). The 390-nt G-less transcript is shown. (C) The single-stranded ends of ctDNA inhibit escape commitment. ctDNA was treated with Klenow fragment in the presence of dNTPs to remove 5′ and 3′ single-stranded overhangs and was subsequently added to assays at the points indicated. The 390-nt G-less transcript is shown.

    Journal: Molecular and Cellular Biology

    Article Title: Translocation after Synthesis of a Four-Nucleotide RNA Commits RNA Polymerase II to Promoter Escape

    doi: 10.1128/MCB.22.3.762-773.2002

    Figure Lengend Snippet: Single-stranded nucleic acid ends are required to inhibit escape commitment. (A) Method used to monitor escape commitment. Nucleic acid inhibitors were added to transcription reaction mixtures at points 1, 2, and 3. (B) Poly(dI-dC) does not inhibit escape commitment. Poly(dI-dC) and ctDNA were added to transcription reactions at the points indicated (see panel A for the method). The 390-nt G-less transcript is shown. (C) The single-stranded ends of ctDNA inhibit escape commitment. ctDNA was treated with Klenow fragment in the presence of dNTPs to remove 5′ and 3′ single-stranded overhangs and was subsequently added to assays at the points indicated. The 390-nt G-less transcript is shown.

    Article Snippet: For the experiment for which results are shown in Fig. , ctDNA was treated with Klenow fragment (Promega) at a final concentration of 1 U/μg in a buffer consisting of 50 mM Tris · HCl (pH 7.2), 10 mM MgSO4 , 0.1 mM dithiothreitol (DTT), 20 μg of bovine serum albumin/ml, and 40 μM each dATP, dCTP, dGTP, and dTTP.

    Techniques: