t4 dna polymerase  (TaKaRa)

 
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    Name:
    T4 DNA Polymerase
    Description:
    T4 DNA Polymerase catalyzes the 5 →3 incorporation of nucleotides into double stranded DNA using single stranded and primed DNA as a template It possesses strong 3 →5 exonuclease proofreading activity but does not exhibit 5 →3 exonuclease activity Typical T4 DNA polymerase protocols include 5 DNA overhang blunting as well as the generation of blunt double stranded DNA from double stranded DNA containing 3 overhangs These DNA products are often used in blunt cloning T4 DNA Polymerase is supplied in a buffer of 200 mM potassium phosphate pH 6 5 10 mM DTT and 50 glycerol
    Catalog Number:
    2040b
    Price:
    None
    Size:
    500 Units
    Category:
    T4 DNA Polymerase DNA polymerases Modifying enzymes Cloning
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    Structured Review

    TaKaRa t4 dna polymerase
    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or <t>T4</t> DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    T4 DNA Polymerase catalyzes the 5 →3 incorporation of nucleotides into double stranded DNA using single stranded and primed DNA as a template It possesses strong 3 →5 exonuclease proofreading activity but does not exhibit 5 →3 exonuclease activity Typical T4 DNA polymerase protocols include 5 DNA overhang blunting as well as the generation of blunt double stranded DNA from double stranded DNA containing 3 overhangs These DNA products are often used in blunt cloning T4 DNA Polymerase is supplied in a buffer of 200 mM potassium phosphate pH 6 5 10 mM DTT and 50 glycerol
    https://www.bioz.com/result/t4 dna polymerase/product/TaKaRa
    Average 95 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-08
    95/100 stars

    Images

    1) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    Figure Legend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Techniques Used: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    2) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    Figure Legend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Techniques Used: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    3) Product Images from "A novel and simple method for construction of recombinant adenoviruses"

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl449

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
    Figure Legend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Techniques Used: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

    4) Product Images from "Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase"

    Article Title: Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsy018

    G-tailed DNA end efficiently acquired GAOs of different lengths. G-tailed FAM70 DNA (G4 80% G5 20% ) was reacted with GAOs SA725 to SA732 of different lengths for 5 min and then purified using DNA purification columns. The purification product was further subjected to blunting by T4 DNA polymerase and then analysed. Asterisks indicate the concatenated products, and the arrow indicates the peaks of the original size (70 nucleotides).
    Figure Legend Snippet: G-tailed DNA end efficiently acquired GAOs of different lengths. G-tailed FAM70 DNA (G4 80% G5 20% ) was reacted with GAOs SA725 to SA732 of different lengths for 5 min and then purified using DNA purification columns. The purification product was further subjected to blunting by T4 DNA polymerase and then analysed. Asterisks indicate the concatenated products, and the arrow indicates the peaks of the original size (70 nucleotides).

    Techniques Used: Purification, DNA Purification

    5) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    Figure Legend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Techniques Used: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    6) Product Images from "Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase"

    Article Title: Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsy018

    G-tailed DNA end efficiently acquired GAOs of different lengths. G-tailed FAM70 DNA (G4 80% G5 20% ) was reacted with GAOs SA725 to SA732 of different lengths for 5 min and then purified using DNA purification columns. The purification product was further subjected to blunting by T4 DNA polymerase and then analysed. Asterisks indicate the concatenated products, and the arrow indicates the peaks of the original size (70 nucleotides).
    Figure Legend Snippet: G-tailed DNA end efficiently acquired GAOs of different lengths. G-tailed FAM70 DNA (G4 80% G5 20% ) was reacted with GAOs SA725 to SA732 of different lengths for 5 min and then purified using DNA purification columns. The purification product was further subjected to blunting by T4 DNA polymerase and then analysed. Asterisks indicate the concatenated products, and the arrow indicates the peaks of the original size (70 nucleotides).

    Techniques Used: Purification, DNA Purification

    7) Product Images from "A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses"

    Article Title: A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

    Journal: Scientific Reports

    doi: 10.1038/srep06438

    Absolute quantification of Tomato yellow leaf curl Sardinia virus (TYLCSV) virion-sense (VS) and complementary-sense (CS) ssDNA molecules, in the presence of an equimolar amount of dsDNA molecules of the same virus. Circular ssDNA molecules bearing the TYLCSV VS strand (10 6 copies) were mixed with the same quantity of circular ssDNA bearing the TYLCSV CS strand and with 10 6 molecules of phagemid dsDNA carrying the TYLCSV genome. The mix was denatured and used as template for T4 DNA polymerase primer extension with the primers described in Fig. 1 , followed by qPCR with the indicated primer combinations for quantification of VS strands (grey bar), CS strands (white bar) and total viral DNA (VS and CS strands) (black bar). Data represent the average of three technical qPCR replicates.
    Figure Legend Snippet: Absolute quantification of Tomato yellow leaf curl Sardinia virus (TYLCSV) virion-sense (VS) and complementary-sense (CS) ssDNA molecules, in the presence of an equimolar amount of dsDNA molecules of the same virus. Circular ssDNA molecules bearing the TYLCSV VS strand (10 6 copies) were mixed with the same quantity of circular ssDNA bearing the TYLCSV CS strand and with 10 6 molecules of phagemid dsDNA carrying the TYLCSV genome. The mix was denatured and used as template for T4 DNA polymerase primer extension with the primers described in Fig. 1 , followed by qPCR with the indicated primer combinations for quantification of VS strands (grey bar), CS strands (white bar) and total viral DNA (VS and CS strands) (black bar). Data represent the average of three technical qPCR replicates.

    Techniques Used: Real-time Polymerase Chain Reaction

    Schematic representation of a two-step quantitative PCR (qPCR) procedure for the quantification of virion-sense (VS) and complementary-sense (CS) DNA molecules. (A) Amplification of the VS strand using the OCS-TAG primer for T4 DNA polymerase extension and subsequent qPCR amplification with OVS and TAG primers. (B) Amplification of the CS strand using the OVS-TAG primer for T4 DNA polymerase extension followed by qPCR amplification with OCS and TAG primers. (C) qPCR to quantify both VS and CS strands using OVS and OCS primers. Primers used for T4 polymerase extension are removed prior to performing qPCR.
    Figure Legend Snippet: Schematic representation of a two-step quantitative PCR (qPCR) procedure for the quantification of virion-sense (VS) and complementary-sense (CS) DNA molecules. (A) Amplification of the VS strand using the OCS-TAG primer for T4 DNA polymerase extension and subsequent qPCR amplification with OVS and TAG primers. (B) Amplification of the CS strand using the OVS-TAG primer for T4 DNA polymerase extension followed by qPCR amplification with OCS and TAG primers. (C) qPCR to quantify both VS and CS strands using OVS and OCS primers. Primers used for T4 polymerase extension are removed prior to performing qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    8) Product Images from "A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses"

    Article Title: A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

    Journal: Scientific Reports

    doi: 10.1038/srep06438

    Absolute quantification of Tomato yellow leaf curl Sardinia virus (TYLCSV) virion-sense (VS) and complementary-sense (CS) ssDNA molecules, in the presence of an equimolar amount of dsDNA molecules of the same virus. Circular ssDNA molecules bearing the TYLCSV VS strand (10 6 copies) were mixed with the same quantity of circular ssDNA bearing the TYLCSV CS strand and with 10 6 molecules of phagemid dsDNA carrying the TYLCSV genome. The mix was denatured and used as template for T4 DNA polymerase primer extension with the primers described in Fig. 1 , followed by qPCR with the indicated primer combinations for quantification of VS strands (grey bar), CS strands (white bar) and total viral DNA (VS and CS strands) (black bar). Data represent the average of three technical qPCR replicates.
    Figure Legend Snippet: Absolute quantification of Tomato yellow leaf curl Sardinia virus (TYLCSV) virion-sense (VS) and complementary-sense (CS) ssDNA molecules, in the presence of an equimolar amount of dsDNA molecules of the same virus. Circular ssDNA molecules bearing the TYLCSV VS strand (10 6 copies) were mixed with the same quantity of circular ssDNA bearing the TYLCSV CS strand and with 10 6 molecules of phagemid dsDNA carrying the TYLCSV genome. The mix was denatured and used as template for T4 DNA polymerase primer extension with the primers described in Fig. 1 , followed by qPCR with the indicated primer combinations for quantification of VS strands (grey bar), CS strands (white bar) and total viral DNA (VS and CS strands) (black bar). Data represent the average of three technical qPCR replicates.

    Techniques Used: Real-time Polymerase Chain Reaction

    Schematic representation of a two-step quantitative PCR (qPCR) procedure for the quantification of virion-sense (VS) and complementary-sense (CS) DNA molecules. (A) Amplification of the VS strand using the OCS-TAG primer for T4 DNA polymerase extension and subsequent qPCR amplification with OVS and TAG primers. (B) Amplification of the CS strand using the OVS-TAG primer for T4 DNA polymerase extension followed by qPCR amplification with OCS and TAG primers. (C) qPCR to quantify both VS and CS strands using OVS and OCS primers. Primers used for T4 polymerase extension are removed prior to performing qPCR.
    Figure Legend Snippet: Schematic representation of a two-step quantitative PCR (qPCR) procedure for the quantification of virion-sense (VS) and complementary-sense (CS) DNA molecules. (A) Amplification of the VS strand using the OCS-TAG primer for T4 DNA polymerase extension and subsequent qPCR amplification with OVS and TAG primers. (B) Amplification of the CS strand using the OVS-TAG primer for T4 DNA polymerase extension followed by qPCR amplification with OCS and TAG primers. (C) qPCR to quantify both VS and CS strands using OVS and OCS primers. Primers used for T4 polymerase extension are removed prior to performing qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    9) Product Images from "Effects of acetaldehyde-induced DNA lesions on DNA metabolism"

    Article Title: Effects of acetaldehyde-induced DNA lesions on DNA metabolism

    Journal: Genes and Environment

    doi: 10.1186/s41021-019-0142-7

    Exonucleolytic digestion in acetaldehyde-treated oligonucleotides. a Schematic drawing of 32 P -labelled 70-mer oligonucleotide. b Action of the exonuclease function of T4 DNA polymerase on an oligonucleotide containing acetaldehyde induced DNA lesions. Non-treated oligonucleotides (lanes 1–5) and acetaldehyde-treated oligonucleotides (lanes 6–10) were digested with increasing amounts of T4 DNA polymerase (0, 0.3, 0.75, 1.5, and 3 units) in the absence of deoxynucleoside triphosphates at 37 °C for 30 min
    Figure Legend Snippet: Exonucleolytic digestion in acetaldehyde-treated oligonucleotides. a Schematic drawing of 32 P -labelled 70-mer oligonucleotide. b Action of the exonuclease function of T4 DNA polymerase on an oligonucleotide containing acetaldehyde induced DNA lesions. Non-treated oligonucleotides (lanes 1–5) and acetaldehyde-treated oligonucleotides (lanes 6–10) were digested with increasing amounts of T4 DNA polymerase (0, 0.3, 0.75, 1.5, and 3 units) in the absence of deoxynucleoside triphosphates at 37 °C for 30 min

    Techniques Used:

    10) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    Figure Legend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Techniques Used: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    11) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    Figure Legend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Techniques Used: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    12) Product Images from "A novel and simple method for construction of recombinant adenoviruses"

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl449

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
    Figure Legend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Techniques Used: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

    13) Product Images from "I-ApeI: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1"

    Article Title: I-ApeI: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni118

    Recognition sequence and CS of I-ApeI. ( A ) Summary of recognition sequence mapping. The numbering below the sequence is described in the text. The region in which deletions and substitutions abolished cleavage is shaded in gray. The IS of the ApeK1.S908 intron is indicated by the arrow. ( B ) Sequences (top strand) of the recognition site mutants. WT refers to NcoI-digested pWT. The name of each deletion mutant is given on the left. Nucleotides replacing the deleted regions are represented in gray lower case. ( C ) I-ApeI cleavage assay for recognition site mutants. Each substrate (0.1 pmol) was incubated for 10 min at 90°C with His 6 I–ApeI (10 pmol). The symbols are as described in Figure 1D . ( D ) CS mapping. The products of I-ApeI cleavage reactions (lane X) were subjected to electrophoresis alongside sequencing ladders. The CSs (open arrowheads) on the top strand (left panel) and bottom strand (right panel) are shown. Sequencing lanes A, C, G and T are denoted by the dideoxynucleotide species used in the reaction. The sequence of the target DNA immediately flanking the CS on each strand is shown on the right. ( E ) Determination of the cohesive termini generated by I-ApeI cleavage. The sequencing chromatogram for the area of interest on the bottom strand of pWTΔPE8 is shown. The gray arrowhead denotes the junction of the blunt ends produced by T4 DNA polymerase action on the I-ApeI digest.
    Figure Legend Snippet: Recognition sequence and CS of I-ApeI. ( A ) Summary of recognition sequence mapping. The numbering below the sequence is described in the text. The region in which deletions and substitutions abolished cleavage is shaded in gray. The IS of the ApeK1.S908 intron is indicated by the arrow. ( B ) Sequences (top strand) of the recognition site mutants. WT refers to NcoI-digested pWT. The name of each deletion mutant is given on the left. Nucleotides replacing the deleted regions are represented in gray lower case. ( C ) I-ApeI cleavage assay for recognition site mutants. Each substrate (0.1 pmol) was incubated for 10 min at 90°C with His 6 I–ApeI (10 pmol). The symbols are as described in Figure 1D . ( D ) CS mapping. The products of I-ApeI cleavage reactions (lane X) were subjected to electrophoresis alongside sequencing ladders. The CSs (open arrowheads) on the top strand (left panel) and bottom strand (right panel) are shown. Sequencing lanes A, C, G and T are denoted by the dideoxynucleotide species used in the reaction. The sequence of the target DNA immediately flanking the CS on each strand is shown on the right. ( E ) Determination of the cohesive termini generated by I-ApeI cleavage. The sequencing chromatogram for the area of interest on the bottom strand of pWTΔPE8 is shown. The gray arrowhead denotes the junction of the blunt ends produced by T4 DNA polymerase action on the I-ApeI digest.

    Techniques Used: Sequencing, Mutagenesis, Cleavage Assay, Incubation, Electrophoresis, Generated, Produced

    14) Product Images from "I-ApeI: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1"

    Article Title: I-ApeI: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni118

    Recognition sequence and CS of I-ApeI. ( A ) Summary of recognition sequence mapping. The numbering below the sequence is described in the text. The region in which deletions and substitutions abolished cleavage is shaded in gray. The IS of the ApeK1.S908 intron is indicated by the arrow. ( B ) Sequences (top strand) of the recognition site mutants. WT refers to NcoI-digested pWT. The name of each deletion mutant is given on the left. Nucleotides replacing the deleted regions are represented in gray lower case. ( C ) I-ApeI cleavage assay for recognition site mutants. Each substrate (0.1 pmol) was incubated for 10 min at 90°C with His 6 I–ApeI (10 pmol). The symbols are as described in Figure 1D . ( D ) CS mapping. The products of I-ApeI cleavage reactions (lane X) were subjected to electrophoresis alongside sequencing ladders. The CSs (open arrowheads) on the top strand (left panel) and bottom strand (right panel) are shown. Sequencing lanes A, C, G and T are denoted by the dideoxynucleotide species used in the reaction. The sequence of the target DNA immediately flanking the CS on each strand is shown on the right. ( E ) Determination of the cohesive termini generated by I-ApeI cleavage. The sequencing chromatogram for the area of interest on the bottom strand of pWTΔPE8 is shown. The gray arrowhead denotes the junction of the blunt ends produced by T4 DNA polymerase action on the I-ApeI digest.
    Figure Legend Snippet: Recognition sequence and CS of I-ApeI. ( A ) Summary of recognition sequence mapping. The numbering below the sequence is described in the text. The region in which deletions and substitutions abolished cleavage is shaded in gray. The IS of the ApeK1.S908 intron is indicated by the arrow. ( B ) Sequences (top strand) of the recognition site mutants. WT refers to NcoI-digested pWT. The name of each deletion mutant is given on the left. Nucleotides replacing the deleted regions are represented in gray lower case. ( C ) I-ApeI cleavage assay for recognition site mutants. Each substrate (0.1 pmol) was incubated for 10 min at 90°C with His 6 I–ApeI (10 pmol). The symbols are as described in Figure 1D . ( D ) CS mapping. The products of I-ApeI cleavage reactions (lane X) were subjected to electrophoresis alongside sequencing ladders. The CSs (open arrowheads) on the top strand (left panel) and bottom strand (right panel) are shown. Sequencing lanes A, C, G and T are denoted by the dideoxynucleotide species used in the reaction. The sequence of the target DNA immediately flanking the CS on each strand is shown on the right. ( E ) Determination of the cohesive termini generated by I-ApeI cleavage. The sequencing chromatogram for the area of interest on the bottom strand of pWTΔPE8 is shown. The gray arrowhead denotes the junction of the blunt ends produced by T4 DNA polymerase action on the I-ApeI digest.

    Techniques Used: Sequencing, Mutagenesis, Cleavage Assay, Incubation, Electrophoresis, Generated, Produced

    Related Articles

    Clone Assay:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. After the unidirectionally reassembled DNA fragments were purified, they were blunt-ended on both ends with T4 DNA polymerase or S1 nuclease and then sticky-ended on the 3′ end with Sal I. Fragments in the range of about 500 to 960 bp were isolated by preparative agarose gel electrophoresis and cloned into pSTV28. .. As shown below, the treatment of T4 DNA polymerase and the subsequent restriction digestion guaranteed that the randomly truncated and shuffled DNA fragments having exclusively MURA primer sequences at their 3′ end (i.e., fragments reassembled together with MURA primer) could be correctly ligated into 5′ blunt- and 3′ sticky-ended vector (i.e., Sma I- and Sal I-digested pSTV28).

    Article Title: A novel and simple method for construction of recombinant adenoviruses
    Article Snippet: .. Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR. ..

    Amplification:

    Article Title: A novel and simple method for construction of recombinant adenoviruses
    Article Snippet: .. Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR. ..

    Agarose Gel Electrophoresis:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. After the unidirectionally reassembled DNA fragments were purified, they were blunt-ended on both ends with T4 DNA polymerase or S1 nuclease and then sticky-ended on the 3′ end with Sal I. Fragments in the range of about 500 to 960 bp were isolated by preparative agarose gel electrophoresis and cloned into pSTV28. .. As shown below, the treatment of T4 DNA polymerase and the subsequent restriction digestion guaranteed that the randomly truncated and shuffled DNA fragments having exclusively MURA primer sequences at their 3′ end (i.e., fragments reassembled together with MURA primer) could be correctly ligated into 5′ blunt- and 3′ sticky-ended vector (i.e., Sma I- and Sal I-digested pSTV28).

    Isolation:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. After the unidirectionally reassembled DNA fragments were purified, they were blunt-ended on both ends with T4 DNA polymerase or S1 nuclease and then sticky-ended on the 3′ end with Sal I. Fragments in the range of about 500 to 960 bp were isolated by preparative agarose gel electrophoresis and cloned into pSTV28. .. As shown below, the treatment of T4 DNA polymerase and the subsequent restriction digestion guaranteed that the randomly truncated and shuffled DNA fragments having exclusively MURA primer sequences at their 3′ end (i.e., fragments reassembled together with MURA primer) could be correctly ligated into 5′ blunt- and 3′ sticky-ended vector (i.e., Sma I- and Sal I-digested pSTV28).

    Generated:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. When the plasmids from randomly selected members were digested with Eco RI and Sal I, we determined that the MURA products were correctly introduced in the form of 5′ blunt- and 3′ sticky-ended fragments, which had been generated by treating T4 DNA polymerase and restriction enzyme Sal I. ..

    Purification:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. The MURA reaction was performed with an automatic thermal cycler (Ericomp, San Diego, Calif.) for 30 cycles, with each cycle consisting of 94°C for 60 s, 54 to 62°C for 40 s, and 72°C for 60 s. The MURA products were purified with a Qiaquick PCR purification kit (Qiagen) and then treated with 1 U of T4 DNA polymerase (Takara) in 40 μl of the manufacturer's 1X buffer and 0.17 mM each dNTP for 10 min at 37°C or with 10 U of S1 nuclease (Takara) in 40 μl of the manufacturer's 1X buffer for 10 min at 25°C. .. After being purified with a Qiaquick PCR purification kit, the blunt-ended MURA products were digested with Sal I for the purpose of converting the blunt ends to sticky ends at the 3′ ends of the MURA products.

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. After the unidirectionally reassembled DNA fragments were purified, they were blunt-ended on both ends with T4 DNA polymerase or S1 nuclease and then sticky-ended on the 3′ end with Sal I. Fragments in the range of about 500 to 960 bp were isolated by preparative agarose gel electrophoresis and cloned into pSTV28. .. As shown below, the treatment of T4 DNA polymerase and the subsequent restriction digestion guaranteed that the randomly truncated and shuffled DNA fragments having exclusively MURA primer sequences at their 3′ end (i.e., fragments reassembled together with MURA primer) could be correctly ligated into 5′ blunt- and 3′ sticky-ended vector (i.e., Sma I- and Sal I-digested pSTV28).

    Incubation:

    Article Title: A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses
    Article Snippet: .. Reaction mixes were denatured (except where indicated) at 95°C for 10 minutes, cooled down to room temperature, and incubated at 37°C for 30 minutes with 1 unit of T4 DNA polymerase. .. Following the reaction, primers were removed using the QIAquick purification kit (QIAGEN, Hamburg, Germany) according to the manufacturer's instructions, and DNA eluted in 50 μl of water.

    other:

    Article Title: Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase
    Article Snippet: T4 DNA polymerase was purchased from Takara (Japan).

    Polymerase Chain Reaction:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: .. The MURA reaction was performed with an automatic thermal cycler (Ericomp, San Diego, Calif.) for 30 cycles, with each cycle consisting of 94°C for 60 s, 54 to 62°C for 40 s, and 72°C for 60 s. The MURA products were purified with a Qiaquick PCR purification kit (Qiagen) and then treated with 1 U of T4 DNA polymerase (Takara) in 40 μl of the manufacturer's 1X buffer and 0.17 mM each dNTP for 10 min at 37°C or with 10 U of S1 nuclease (Takara) in 40 μl of the manufacturer's 1X buffer for 10 min at 25°C. .. After being purified with a Qiaquick PCR purification kit, the blunt-ended MURA products were digested with Sal I for the purpose of converting the blunt ends to sticky ends at the 3′ ends of the MURA products.

    Article Title: A novel and simple method for construction of recombinant adenoviruses
    Article Snippet: .. Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR. ..

    Plasmid Preparation:

    Article Title: A novel and simple method for construction of recombinant adenoviruses
    Article Snippet: .. Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR. ..

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    TaKaRa qrt pcr dnase i treated rna
    PPARγ promoter usage in the DL and Min pigs. ( A ) The assembled exon structures of PPARγ in pigs. Peaks show <t>RNA-seq</t> reads mapped to genomic regions in each pig breed at 4 weeks (4W) and 16 weeks (16W) of age ( n = 3 biological replicates). ( B ) Read number mapped to the first exon of each PPARγ isoform. Read number was normalized by reads per million reads (RPM). ( C ) PPARγ promoter usages. PPARγ transcript isoform proportions were calculated by dividing the read number mapped to each first exon by the total read number mapped to all three first exons. ( D ) Expression level verification by <t>qRT-PCR.</t> Blue arrows in A give the exon regions used to design the qRT-PCR primers. Data presented with standard error bars per group ( n = 3).
    Qrt Pcr Dnase I Treated Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa t4 dna polymerase
    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or <t>T4</t> DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.
    T4 Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/TaKaRa
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    Image Search Results


    PPARγ promoter usage in the DL and Min pigs. ( A ) The assembled exon structures of PPARγ in pigs. Peaks show RNA-seq reads mapped to genomic regions in each pig breed at 4 weeks (4W) and 16 weeks (16W) of age ( n = 3 biological replicates). ( B ) Read number mapped to the first exon of each PPARγ isoform. Read number was normalized by reads per million reads (RPM). ( C ) PPARγ promoter usages. PPARγ transcript isoform proportions were calculated by dividing the read number mapped to each first exon by the total read number mapped to all three first exons. ( D ) Expression level verification by qRT-PCR. Blue arrows in A give the exon regions used to design the qRT-PCR primers. Data presented with standard error bars per group ( n = 3).

    Journal: International Journal of Molecular Sciences

    Article Title: Distinct Patterns of PPARγ Promoter Usage, Lipid Degradation Activity, and Gene Expression in Subcutaneous Adipose Tissue of Lean and Obese Swine

    doi: 10.3390/ijms19123892

    Figure Lengend Snippet: PPARγ promoter usage in the DL and Min pigs. ( A ) The assembled exon structures of PPARγ in pigs. Peaks show RNA-seq reads mapped to genomic regions in each pig breed at 4 weeks (4W) and 16 weeks (16W) of age ( n = 3 biological replicates). ( B ) Read number mapped to the first exon of each PPARγ isoform. Read number was normalized by reads per million reads (RPM). ( C ) PPARγ promoter usages. PPARγ transcript isoform proportions were calculated by dividing the read number mapped to each first exon by the total read number mapped to all three first exons. ( D ) Expression level verification by qRT-PCR. Blue arrows in A give the exon regions used to design the qRT-PCR primers. Data presented with standard error bars per group ( n = 3).

    Article Snippet: 4.4. qRT-PCR DNase I-treated RNA was reverse transcribed using SuperScript III and oligo-dT primers. cDNA was analyzed by qRT-PCR using SYBR Premix Ex Taq (Takara, Otsu, Shiga, Japan).

    Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR

    Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Journal: Applied and Environmental Microbiology

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Figure Lengend Snippet: Schematic overview of the construction of a DNA-shuffled and truncated enzyme library by the MURA method. The sequences of the MURA primer can be chosen on any desired site of the parent gene. This figure represents a procedure using a 3′-complementary MURA primer, and thus N-terminal-truncated and shuffled variants of PlaA are constructed. The steps shown are random fragmentation of the parent gene pool by DNase I (a), unidirectional reassembly with the MURA primer (b), separation of the fragments of interest by preparative agarose gel electrophoresis (c), formation of blunt ends by S1 nuclease or T4 DNA polymerase on both termini (d), sticky-end formation by a restriction enzyme on one terminus (e), and ligation into an expression vector (f). The order of steps c, d, and e can be altered to d, e, and c without affecting the efficiency of library construction. The diamonds on the bars representing genes indicate possible mutations during shuffled and unidirectional reassembly PCR.

    Article Snippet: After the unidirectionally reassembled DNA fragments were purified, they were blunt-ended on both ends with T4 DNA polymerase or S1 nuclease and then sticky-ended on the 3′ end with Sal I. Fragments in the range of about 500 to 960 bp were isolated by preparative agarose gel electrophoresis and cloned into pSTV28.

    Techniques: Construct, Agarose Gel Electrophoresis, Ligation, Expressing, Plasmid Preparation, Polymerase Chain Reaction

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Journal: Nucleic Acids Research

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    doi: 10.1093/nar/gkl449

    Figure Lengend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Article Snippet: Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR.

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay