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Promega taq polymerase
PAGE gel image of the ethidium bromide stained and UV visualised 98 nt <t>PCR</t> fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for <t>Taq</t> polymerase during the PCR reaction but 8c is not.
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Images

1) Product Images from "Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity"

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

2) Product Images from "Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity"

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

3) Product Images from "Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity"

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

4) Product Images from "Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR"

Article Title: Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.
Figure Legend Snippet: PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Derivative Assay, Modification, Molecular Weight

5) Product Images from "Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity"

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

6) Product Images from "Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR"

Article Title: Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.
Figure Legend Snippet: PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Derivative Assay, Modification, Molecular Weight

7) Product Images from "Intrastrain and interstrain genetic variation within a paralogous gene family in Chlamydia pneumoniae"

Article Title: Intrastrain and interstrain genetic variation within a paralogous gene family in Chlamydia pneumoniae

Journal: BMC Microbiology

doi: 10.1186/1471-2180-2-38

Variation of the homopolymeric cytosine (polyC) tract in the Cpn 1054 gene family in C. pneumoniae . In each panel the vertical axis indicates the number of individual clones with a specific length of polyC. The horizontal axis indicates the number of cytosines within an individual polyC tract. (A) Intrastrain variation of the length of the polyC tracts of Cpn 043, 1054,1055 within strain AR39. (B) Variation of the length of the polyC tract in Cpn 1055 within AR39, AR 458 and PS32. (C) The variation of the length of the poly C tract within Cpn 1055 of AR39 identified in recombinant clones using Taq and Pwo DNA polymerases.
Figure Legend Snippet: Variation of the homopolymeric cytosine (polyC) tract in the Cpn 1054 gene family in C. pneumoniae . In each panel the vertical axis indicates the number of individual clones with a specific length of polyC. The horizontal axis indicates the number of cytosines within an individual polyC tract. (A) Intrastrain variation of the length of the polyC tracts of Cpn 043, 1054,1055 within strain AR39. (B) Variation of the length of the polyC tract in Cpn 1055 within AR39, AR 458 and PS32. (C) The variation of the length of the poly C tract within Cpn 1055 of AR39 identified in recombinant clones using Taq and Pwo DNA polymerases.

Techniques Used: Clone Assay, Recombinant

8) Product Images from "Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood quantitative, rapid identification of active Ebola virus infection in unprocessed blood †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03281a"

Article Title: Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood quantitative, rapid identification of active Ebola virus infection in unprocessed blood †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03281a

Journal: Chemical Science

doi: 10.1039/c7sc03281a

Overcoming the dose-dependent inhibitory effect of blood on qRT-PCR. Increasing amounts of fresh, unprocessed human blood introduced into qRT-PCR reactions (0.0–5.0% v/v final blood concentration) result in comparable dose-dependent inhibition of FAM-labelled Taqman® probe-based RT-PCR fluorogenicity (amplification plateau) whether the Promega hot-start wild type Taq polymerase (a) or the blood-optimised OmniTaq 2 polymerase (b) is used in conjunction with an MMuLV reverse transcriptase to detect 4 × 10 8 copies of MS2 coliphage RNA. Reaction progression is reported for each of two technical replicates in relative fluorescence units (RFU) normalised to no template control (–ve c) against PCR cycle value. Far red/infrared fluorescent dyes (c) and dye–quencher combinations (d) were screened by spectrophotometry under 620 nm laser diode simulation for utility in blood-containing reactions. The impact of blood on dye fluorogenicity is expressed as the change (Δ F N ) in background-subtracted fluorescence emission (RFU) in the presence and absence of human blood (mean of two technical replicates). DDB: deep dark blue; VRH: Viridian Red High; VRL: Viridian Red Low; IWB: Iowa Black; BHQ: Black Hole Quencher.
Figure Legend Snippet: Overcoming the dose-dependent inhibitory effect of blood on qRT-PCR. Increasing amounts of fresh, unprocessed human blood introduced into qRT-PCR reactions (0.0–5.0% v/v final blood concentration) result in comparable dose-dependent inhibition of FAM-labelled Taqman® probe-based RT-PCR fluorogenicity (amplification plateau) whether the Promega hot-start wild type Taq polymerase (a) or the blood-optimised OmniTaq 2 polymerase (b) is used in conjunction with an MMuLV reverse transcriptase to detect 4 × 10 8 copies of MS2 coliphage RNA. Reaction progression is reported for each of two technical replicates in relative fluorescence units (RFU) normalised to no template control (–ve c) against PCR cycle value. Far red/infrared fluorescent dyes (c) and dye–quencher combinations (d) were screened by spectrophotometry under 620 nm laser diode simulation for utility in blood-containing reactions. The impact of blood on dye fluorogenicity is expressed as the change (Δ F N ) in background-subtracted fluorescence emission (RFU) in the presence and absence of human blood (mean of two technical replicates). DDB: deep dark blue; VRH: Viridian Red High; VRL: Viridian Red Low; IWB: Iowa Black; BHQ: Black Hole Quencher.

Techniques Used: Quantitative RT-PCR, Concentration Assay, Inhibition, Reverse Transcription Polymerase Chain Reaction, Amplification, Fluorescence, Polymerase Chain Reaction, Spectrophotometry

9) Product Images from "Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity"

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.
Figure Legend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

10) Product Images from "Identification of a novel metastasis inducing lncRNA which suppresses the KAI1/CD82 metastasis suppressor gene and is upregulated in triple-negative breast cancer"

Article Title: Identification of a novel metastasis inducing lncRNA which suppresses the KAI1/CD82 metastasis suppressor gene and is upregulated in triple-negative breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.18733

KAI1 as-lncRNA; analysis via RT-PCR (A) Products obtained in the presence of actinomycin D in the RTase reaction. Total MDA-MB-231 RNA has been reverse transcribed with M-MLV RTase with/without 50ng/μl Actinomycin D, followed by PCR amplification after dilution of Actinomycin D to 2.5 ng/μl (5/100) or 1.25ng/μl (5/200). (B) Dependence on particular RTase? Total MDA-MB-231 RNA was reverse transcribed with either M-MLV RTase or AMV RTase, and a transcript specific RT-primer (upper) or hexamers (lower), followed by PCR with Hot start Taq-Polymerase. (C) 5’ end mapping. MDA-MB-231 RNA treated by DNase and periodate was reverse transcribed with specific RT5 primer (+RT). PCR amplification led to 705 bp product. (D) 3’ end polyadenylation? Polyadenylated MDA-MB-231 RNA was pulled down with magnetic beads, while polyA minus-RNA remained in the flow through fraction. Both fractions and total RNA were reverse transcribed with KAI1 as-lncRNA specific primer. PCR amplifies a 168 bp product. (E) nuclear localization MDA-MB-231 RNA (2ug nuclear or total RNA fraction) was reverse transcribed with KAI1 as-lncRNA specific primer in presence (+RT) or absence (−RT) of RTase. 317 bp amplified PCR product indicated with an arrow. Abbreviations: –RT= no RT enzyme. –Primer/ -Pri (+)= no primer in RT reaction. –PrN = no primer in nuclear RNA fraction; -PrT=no primer in total RNA fraction. +RT= with RT enzyme. –tpl= no template. +ActD= 50ng/ μl Actinomycin D in RT reaction. 100 bp= DNA ladder. M= DNA marker; gnmc= genomic DNA.
Figure Legend Snippet: KAI1 as-lncRNA; analysis via RT-PCR (A) Products obtained in the presence of actinomycin D in the RTase reaction. Total MDA-MB-231 RNA has been reverse transcribed with M-MLV RTase with/without 50ng/μl Actinomycin D, followed by PCR amplification after dilution of Actinomycin D to 2.5 ng/μl (5/100) or 1.25ng/μl (5/200). (B) Dependence on particular RTase? Total MDA-MB-231 RNA was reverse transcribed with either M-MLV RTase or AMV RTase, and a transcript specific RT-primer (upper) or hexamers (lower), followed by PCR with Hot start Taq-Polymerase. (C) 5’ end mapping. MDA-MB-231 RNA treated by DNase and periodate was reverse transcribed with specific RT5 primer (+RT). PCR amplification led to 705 bp product. (D) 3’ end polyadenylation? Polyadenylated MDA-MB-231 RNA was pulled down with magnetic beads, while polyA minus-RNA remained in the flow through fraction. Both fractions and total RNA were reverse transcribed with KAI1 as-lncRNA specific primer. PCR amplifies a 168 bp product. (E) nuclear localization MDA-MB-231 RNA (2ug nuclear or total RNA fraction) was reverse transcribed with KAI1 as-lncRNA specific primer in presence (+RT) or absence (−RT) of RTase. 317 bp amplified PCR product indicated with an arrow. Abbreviations: –RT= no RT enzyme. –Primer/ -Pri (+)= no primer in RT reaction. –PrN = no primer in nuclear RNA fraction; -PrT=no primer in total RNA fraction. +RT= with RT enzyme. –tpl= no template. +ActD= 50ng/ μl Actinomycin D in RT reaction. 100 bp= DNA ladder. M= DNA marker; gnmc= genomic DNA.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Polymerase Chain Reaction, Amplification, Magnetic Beads, Flow Cytometry, Marker

11) Product Images from "7-Deaza-2`-deoxyguanosine allows PCR and sequencing reactions from CpG islands"

Article Title: 7-Deaza-2`-deoxyguanosine allows PCR and sequencing reactions from CpG islands

Journal: Molecular Pathology

doi:

(A) 7-Deaza-2`-deoxyguanosine (deaza-dGTP) allows PCR of the human p16 INK4A promoter because the specific 140 bp product (black arrow, 140) is clearly visible whether standard Taq polymerase (no hot start) or AmpliTaq Gold (hot start) is used. (B) dGTP gives rise to unspecific byproducts (black arrow, by), which are reduced if hot start PCR is performed. The 600 bp band of the 100 bp ladder standard is indicated (white arrow, 600).
Figure Legend Snippet: (A) 7-Deaza-2`-deoxyguanosine (deaza-dGTP) allows PCR of the human p16 INK4A promoter because the specific 140 bp product (black arrow, 140) is clearly visible whether standard Taq polymerase (no hot start) or AmpliTaq Gold (hot start) is used. (B) dGTP gives rise to unspecific byproducts (black arrow, by), which are reduced if hot start PCR is performed. The 600 bp band of the 100 bp ladder standard is indicated (white arrow, 600).

Techniques Used: Polymerase Chain Reaction, Hot Start PCR

12) Product Images from "Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR"

Article Title: Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

Journal: Nucleic Acids Research

doi:

PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.
Figure Legend Snippet: PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Derivative Assay, Modification, Molecular Weight

Related Articles

Amplification:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. A decrease of 4-fold in slippage peak was observed in DNA obtained from processive amplification using Taq DNA pol/TBD compared with Taq DNA polymerase. ..

Ligation:

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'
Article Snippet: .. A typical 10 µl ligation reaction contained 250 ng of DNA fragments, 5 µM Taq I adapters, 25 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 5 mM dithiothreitol, 500 µΜ ATP, 12.5 µg/ml BSA and 200 cohesive-end units of T4 DNA ligase (New England Biolabs) and was incubated at 16°C for 12–16 h. Primary PCR was done in a 25 µl reaction volume containing 200 µM each dNTP, 40 nM each primer (Taq1-N 5′-ATGAGTCCTGACCGA, and Tn3-O2, 5′-TTAACGTGAGTTTTCGTTCCACTG), 2 mM MgSO4 and 1 U Taq DNA polymerase (Promega) in 1× PCR buffer (20 mM Tris–HCl pH 9.2, 10 mM KCl, 10 mM ammonium sulfate, 0.1% Triton X-100) and adapter-ligated DNA. .. The amount of adapter-ligated DNA used was 2.5 ng for individual mutants or 250 ng for pooled mutants, added directly from undiluted ligation reaction or from ligation reaction diluted 10-fold in water.

Polymerase Chain Reaction:

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity
Article Snippet: .. Both of the analogues 5a and 5b have been prepared previously and were reported not to be substrates for Taq polymerase during PCR ( , ). ..

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity
Article Snippet: .. Consequently, we have studied the incorporation of the modified triphosphates during PCR reactions mediated by Taq polymerase using template DNAs varying from 62 to 224 nt. .. Whilst other thermostable DNA polymerases ( Tth from Thermus thermophilus and Vent from Thermococcus litoralis ) were investigated, initial studies indicated that the best results for amplification, using both the modified and natural nucleoside triphosphates, were produced using Taq enzyme.

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'
Article Snippet: .. A typical 10 µl ligation reaction contained 250 ng of DNA fragments, 5 µM Taq I adapters, 25 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 5 mM dithiothreitol, 500 µΜ ATP, 12.5 µg/ml BSA and 200 cohesive-end units of T4 DNA ligase (New England Biolabs) and was incubated at 16°C for 12–16 h. Primary PCR was done in a 25 µl reaction volume containing 200 µM each dNTP, 40 nM each primer (Taq1-N 5′-ATGAGTCCTGACCGA, and Tn3-O2, 5′-TTAACGTGAGTTTTCGTTCCACTG), 2 mM MgSO4 and 1 U Taq DNA polymerase (Promega) in 1× PCR buffer (20 mM Tris–HCl pH 9.2, 10 mM KCl, 10 mM ammonium sulfate, 0.1% Triton X-100) and adapter-ligated DNA. .. The amount of adapter-ligated DNA used was 2.5 ng for individual mutants or 250 ng for pooled mutants, added directly from undiluted ligation reaction or from ligation reaction diluted 10-fold in water.

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'
Article Snippet: .. Ten microliters of the enzyme mixture, having 1 U Taq DNA polymerase in 1× PCR buffer, was made separately and overlaid on the wax layer. .. The samples were kept in the thermal cycler (MJ Research Minicycler or Techne Genius) and taken through 30 cycles as follows.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Huang M.M., Arnheim,N. and Goodman,M.F. (1992) Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. ..

Incubation:

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'
Article Snippet: .. A typical 10 µl ligation reaction contained 250 ng of DNA fragments, 5 µM Taq I adapters, 25 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 5 mM dithiothreitol, 500 µΜ ATP, 12.5 µg/ml BSA and 200 cohesive-end units of T4 DNA ligase (New England Biolabs) and was incubated at 16°C for 12–16 h. Primary PCR was done in a 25 µl reaction volume containing 200 µM each dNTP, 40 nM each primer (Taq1-N 5′-ATGAGTCCTGACCGA, and Tn3-O2, 5′-TTAACGTGAGTTTTCGTTCCACTG), 2 mM MgSO4 and 1 U Taq DNA polymerase (Promega) in 1× PCR buffer (20 mM Tris–HCl pH 9.2, 10 mM KCl, 10 mM ammonium sulfate, 0.1% Triton X-100) and adapter-ligated DNA. .. The amount of adapter-ligated DNA used was 2.5 ng for individual mutants or 250 ng for pooled mutants, added directly from undiluted ligation reaction or from ligation reaction diluted 10-fold in water.

other:

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity
Article Snippet: Neither 8b nor 8c were found to be substrates for Taq polymerase, analogous to the parent amino alkane triphosphate ( 8a ).

Activity Assay:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Thioredoxin stimulates the activity of Taq DNA pol/TBD by ∼4-fold and Taq DNA pol/TBD(exo–) enzymatic activities by > 30-fold but had no effect on Taq DNA polymerase. .. Both Taq DNA pol/TBD and Taq DNA pol/TBD(exo–) are as thermostable as the Taq DNA polymerase and Taq DNA polymerase (exo–) enzymes, respectively, following exposure to 94°C for 30 min (Fig. ).

Modification:

Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity
Article Snippet: .. Consequently, we have studied the incorporation of the modified triphosphates during PCR reactions mediated by Taq polymerase using template DNAs varying from 62 to 224 nt. .. Whilst other thermostable DNA polymerases ( Tth from Thermus thermophilus and Vent from Thermococcus litoralis ) were investigated, initial studies indicated that the best results for amplification, using both the modified and natural nucleoside triphosphates, were produced using Taq enzyme.

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    Promega taq polymerase
    PAGE gel image of the ethidium bromide stained and UV visualised 98 nt <t>PCR</t> fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for <t>Taq</t> polymerase during the PCR reaction but 8c is not.
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    PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

    Journal: Nucleic Acids Research

    Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

    doi:

    Figure Lengend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

    Article Snippet: Consequently, we have studied the incorporation of the modified triphosphates during PCR reactions mediated by Taq polymerase using template DNAs varying from 62 to 224 nt.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

    PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

    Journal: Nucleic Acids Research

    Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

    doi:

    Figure Lengend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

    Article Snippet: Consequently, we have studied the incorporation of the modified triphosphates during PCR reactions mediated by Taq polymerase using template DNAs varying from 62 to 224 nt.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

    Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Article Snippet: A decrease of 4-fold in slippage peak was observed in DNA obtained from processive amplification using Taq DNA pol/TBD compared with Taq DNA polymerase.

    Techniques: Sequencing

    Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Article Snippet: A decrease of 4-fold in slippage peak was observed in DNA obtained from processive amplification using Taq DNA pol/TBD compared with Taq DNA polymerase.

    Techniques: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

    Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Article Snippet: A decrease of 4-fold in slippage peak was observed in DNA obtained from processive amplification using Taq DNA pol/TBD compared with Taq DNA polymerase.

    Techniques: Incubation

    The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Article Snippet: A decrease of 4-fold in slippage peak was observed in DNA obtained from processive amplification using Taq DNA pol/TBD compared with Taq DNA polymerase.

    Techniques: Activity Assay, Labeling, Concentration Assay