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  • 88
    TaKaRa 2x takara t4 ligase 9
    2x Takara T4 Ligase 9, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa t4 ligase buffer
    T4 Ligase Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa t4 ligase kit
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    T4 Ligase Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase kit/product/TaKaRa
    Average 99 stars, based on 358 article reviews
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    t4 ligase kit - by Bioz Stars, 2020-05
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    92
    TaKaRa t4 ligase
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    T4 Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 909 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/TaKaRa
    Average 92 stars, based on 909 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-05
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    99
    TaKaRa t4 ligase independent method
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    T4 Ligase Independent Method, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa t4 rna ligase
    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by <t>T4</t> RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.
    T4 Rna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/TaKaRa
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    91
    TaKaRa high fidelity phusion dna polymerase
    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by <t>T4</t> RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.
    High Fidelity Phusion Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: DNA Ligation, Labeling, Incubation, Ligation, Electrophoresis

    Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Labeling, Incubation, Titration, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Incubation, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Ligation, Electrophoresis

    RecA enhances T4 DNA ligase-catalyzed cohesive end- and blunt end-ligation. ( A ) Effects of the amounts of ligase in the presence or absence of RecA. Linear dsDNA with 3′-TGCA four nucleotide overhangs (panel (i)) or 5′-AGCT four nucleotide overhangs (panel (ii)) was incubated with the indicated amounts of T4 DNA ligase, in the presence or absence of RecA, for 40 min in the system without NAD but including ATP. In lanes M, DNA size and marker amounts are the same as in Figure 1B . The plots in panel (iii) show the fractions of residual substrate dsDNA (1L) against the amounts of T4 DNA ligase, quantified from at least three independent experiments. • (closed circles), dsDNA with 3′-four-nucleotide overhangs with RecA; ∘ (open circles), dsDNA with 3′ four-nucleotide overhangs without RecA; ▴ (closed triangles), dsDNA with 5′ four-nucleotide overhangs with RecA; Δ (open triangles), dsDNA with 5′-four-nucleotide overhangs without RecA. ( B ) The effects of RecA on the blunt end-ligation by T4 DNA ligase. Linear dsDNA with blunt ends was incubated with the indicated amounts of T4 DNA ligase, as in A, for 40 min, and the gel profile from a representative experiment is shown in panel (i). In lane M, DNA sizes and marker amounts are the same as in Figure 1B . The plot in panel (ii) was quantified from three independent experiments. ▪ (closed squares), dsDNA with blunt ends with RecA; □ (Open squares), dsDNA with blunt ends without RecA. It should be noted that T4 DNA ligase requires ATP as an essential cofactor, and thus the effects of nucleotide cofactors on the enhancement by RecA could not be tested.

    Journal: Nucleic Acids Research

    Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    doi: 10.1093/nar/gkw998

    Figure Lengend Snippet: RecA enhances T4 DNA ligase-catalyzed cohesive end- and blunt end-ligation. ( A ) Effects of the amounts of ligase in the presence or absence of RecA. Linear dsDNA with 3′-TGCA four nucleotide overhangs (panel (i)) or 5′-AGCT four nucleotide overhangs (panel (ii)) was incubated with the indicated amounts of T4 DNA ligase, in the presence or absence of RecA, for 40 min in the system without NAD but including ATP. In lanes M, DNA size and marker amounts are the same as in Figure 1B . The plots in panel (iii) show the fractions of residual substrate dsDNA (1L) against the amounts of T4 DNA ligase, quantified from at least three independent experiments. • (closed circles), dsDNA with 3′-four-nucleotide overhangs with RecA; ∘ (open circles), dsDNA with 3′ four-nucleotide overhangs without RecA; ▴ (closed triangles), dsDNA with 5′ four-nucleotide overhangs with RecA; Δ (open triangles), dsDNA with 5′-four-nucleotide overhangs without RecA. ( B ) The effects of RecA on the blunt end-ligation by T4 DNA ligase. Linear dsDNA with blunt ends was incubated with the indicated amounts of T4 DNA ligase, as in A, for 40 min, and the gel profile from a representative experiment is shown in panel (i). In lane M, DNA sizes and marker amounts are the same as in Figure 1B . The plot in panel (ii) was quantified from three independent experiments. ▪ (closed squares), dsDNA with blunt ends with RecA; □ (Open squares), dsDNA with blunt ends without RecA. It should be noted that T4 DNA ligase requires ATP as an essential cofactor, and thus the effects of nucleotide cofactors on the enhancement by RecA could not be tested.

    Article Snippet: Escherchia coli and T4 DNA ligases were purchased from Takara Bio Company, Japan.

    Techniques: Ligation, Incubation, Marker

    pAK-TAG expression vector and high level expression of recombinant AK fusion proteins in soluble form. (A) Schematic representation of the pAK-TAG vector. (B) SDS-PAGE analysis of the expression of AK-TNFα, AK-TRAIL, and AK-T4 DNA ligase.

    Journal: PLoS ONE

    Article Title: High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    doi: 10.1371/journal.pone.0156106

    Figure Lengend Snippet: pAK-TAG expression vector and high level expression of recombinant AK fusion proteins in soluble form. (A) Schematic representation of the pAK-TAG vector. (B) SDS-PAGE analysis of the expression of AK-TNFα, AK-TRAIL, and AK-T4 DNA ligase.

    Article Snippet: Chemicals T4 DNA ligase, Taq polymerase, and restriction enzymes were obtained from Takara.

    Techniques: Expressing, Plasmid Preparation, Recombinant, SDS Page

    Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature. The ordinate axis represents the ligation efficiency, which is normalized by that at 16 °C. The standard deviations are obtained from 6 independent experiments.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

    doi: 10.1016/j.bbrep.2016.10.006

    Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature. The ordinate axis represents the ligation efficiency, which is normalized by that at 16 °C. The standard deviations are obtained from 6 independent experiments.

    Article Snippet: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

    Techniques: DNA Ligation, Ligation

    Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 16 °C. The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude. The standard deviations are obtained from 6 independent experiments.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

    doi: 10.1016/j.bbrep.2016.10.006

    Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 16 °C. The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude. The standard deviations are obtained from 6 independent experiments.

    Article Snippet: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

    Techniques: DNA Ligation, Ligation

    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by T4 RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.

    Journal: Nucleic Acids Research

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

    doi: 10.1093/nar/gkl087

    Figure Lengend Snippet: ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by T4 RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.

    Article Snippet: T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO.

    Techniques: In Vitro, Selection, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction