t4 ligase Promega Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs t4 ligase
    PAGE gel demonstrating the generation of a 150-mer fragment after successful adapter ligation. Lanes A 100 bp without ligase, B 100-mer with 1,000 units of T4 ligase, C 100-mer and 25 molar excess adapters with T4 ligase, D dA-tailed 100-mer with T4 ligase, E adapter without ligase, F adapter with T4 ligase, G dA-tailed 100-mer and 25 M excess adapters with 1,000 units of <t>T4</t> ligase
    T4 Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9635 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/New England Biolabs
    Average 99 stars, based on 9635 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher t4 dna ligase
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 25062 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    93
    Promega t4 ligase
    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to <t>T4</t> ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
    T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 2310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/Promega
    Average 93 stars, based on 2310 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    91
    Promega t4 ligase enzyme
    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to <t>T4</t> ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
    T4 Ligase Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase enzyme/product/Promega
    Average 91 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    t4 ligase enzyme - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    92
    Promega t4 ligase buffer
    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to <t>T4</t> ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
    T4 Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase buffer/product/Promega
    Average 92 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    88
    Promega t4 ligase kit
    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to <t>T4</t> ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
    T4 Ligase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase kit/product/Promega
    Average 88 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    t4 ligase kit - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    86
    Promega t4 ligase hc
    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to <t>T4</t> ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
    T4 Ligase Hc, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase hc/product/Promega
    Average 86 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    t4 ligase hc - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    88
    Promega dna t4 ligase
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    Dna T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna t4 ligase/product/Promega
    Average 88 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    dna t4 ligase - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    99
    Promega pgem t easy vector systemwith t4 ligase
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    Pgem T Easy Vector Systemwith T4 Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgem t easy vector systemwith t4 ligase/product/Promega
    Average 99 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    pgem t easy vector systemwith t4 ligase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    92
    Promega t4 dna ligase buffer promega
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    T4 Dna Ligase Buffer Promega, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer promega/product/Promega
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer promega - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    85
    Promega lp25 21 linker
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    Lp25 21 Linker, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lp25 21 linker/product/Promega
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    lp25 21 linker - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    86
    Promega hmgpf pcr amplified gene
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    Hmgpf Pcr Amplified Gene, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hmgpf pcr amplified gene/product/Promega
    Average 86 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    hmgpf pcr amplified gene - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    99
    TaKaRa t4 dna ligase
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/TaKaRa
    Average 99 stars, based on 7380 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    PAGE gel demonstrating the generation of a 150-mer fragment after successful adapter ligation. Lanes A 100 bp without ligase, B 100-mer with 1,000 units of T4 ligase, C 100-mer and 25 molar excess adapters with T4 ligase, D dA-tailed 100-mer with T4 ligase, E adapter without ligase, F adapter with T4 ligase, G dA-tailed 100-mer and 25 M excess adapters with 1,000 units of T4 ligase

    Journal: Analytical and bioanalytical chemistry

    Article Title: Heterobifunctional modification of DNA for conjugation to solid surfaces

    doi: 10.1007/s00216-010-3733-5

    Figure Lengend Snippet: PAGE gel demonstrating the generation of a 150-mer fragment after successful adapter ligation. Lanes A 100 bp without ligase, B 100-mer with 1,000 units of T4 ligase, C 100-mer and 25 molar excess adapters with T4 ligase, D dA-tailed 100-mer with T4 ligase, E adapter without ligase, F adapter with T4 ligase, G dA-tailed 100-mer and 25 M excess adapters with 1,000 units of T4 ligase

    Article Snippet: We observed that, under conditions suggested by the suppliers of the T4 ligase (NEB and Promega), the predominant product was persistently a single-addition product (~100% 125-mer versus ~0% of 150-mer DNA), whereas the yield of conversion was relatively low ( > 70% of original DNA remained unmodified in spite of the 25-fold molar excess of adaptors).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation

    Analysis of PAGE data. a Image intensity profiles are averaged over 20–40 pixels in a given lane and then fitted to multiple Gaussian peaks (using IGOR Pro, Wavemetrics) representing 100-mer-, 125-mer-, and 150-mer-long DNA fragments. Peak areas (normalized by the total fragment length) are used as a quantitative measure of the molar amount of given oligomers. b Time course of changes in the molar fraction of the dA-tailed unmodified DNA (100-mer) and DNA ligated at one end (125-mer) or both ends (150-mer). Six picomoles of 100-mer DNA with 25 molar excess adapters and 400 units of T4 ligase in a total volume of 50 μL were incubated at 16 °C. c Dependence of the conversion of the dA-tailed DNA into end-modified DNA on the concentration of the enzyme for 6-h-long reactions. Six picomoles of 100-mer DNA and 25 molar excess of adapters in a total volume of 10 μL were incubated at 16 °C. Curves in b and c are fits to exponential or double-exponential functions and serve as guides to the eye

    Journal: Analytical and bioanalytical chemistry

    Article Title: Heterobifunctional modification of DNA for conjugation to solid surfaces

    doi: 10.1007/s00216-010-3733-5

    Figure Lengend Snippet: Analysis of PAGE data. a Image intensity profiles are averaged over 20–40 pixels in a given lane and then fitted to multiple Gaussian peaks (using IGOR Pro, Wavemetrics) representing 100-mer-, 125-mer-, and 150-mer-long DNA fragments. Peak areas (normalized by the total fragment length) are used as a quantitative measure of the molar amount of given oligomers. b Time course of changes in the molar fraction of the dA-tailed unmodified DNA (100-mer) and DNA ligated at one end (125-mer) or both ends (150-mer). Six picomoles of 100-mer DNA with 25 molar excess adapters and 400 units of T4 ligase in a total volume of 50 μL were incubated at 16 °C. c Dependence of the conversion of the dA-tailed DNA into end-modified DNA on the concentration of the enzyme for 6-h-long reactions. Six picomoles of 100-mer DNA and 25 molar excess of adapters in a total volume of 10 μL were incubated at 16 °C. Curves in b and c are fits to exponential or double-exponential functions and serve as guides to the eye

    Article Snippet: We observed that, under conditions suggested by the suppliers of the T4 ligase (NEB and Promega), the predominant product was persistently a single-addition product (~100% 125-mer versus ~0% of 150-mer DNA), whereas the yield of conversion was relatively low ( > 70% of original DNA remained unmodified in spite of the 25-fold molar excess of adaptors).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Modification, Concentration Assay

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

    Journal: Journal of Biological Engineering

    Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    doi: 10.1186/1754-1611-1-7

    Figure Lengend Snippet: Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

    Article Snippet: Enzyme amounts: 7.5 units Esp3I, 10 units NheI, 3 weiss units T4 ligase.

    Techniques: Subcloning, Plasmid Preparation, Activity Assay, Marker

    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Journal: Nucleic Acids Research

    Article Title: The role of DNA dependent protein kinase in synapsis of DNA ends

    doi: 10.1093/nar/gkg889

    Figure Lengend Snippet: Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Article Snippet: First, we used AMP-PNP, an ATP analog that supports activity of T4 DNA ligase, but cannot function as a cofactor for DNA-PKCS (Fig. A).

    Techniques: Ligation, Labeling, Incubation, Agarose Gel Electrophoresis