e. coli dna ligase Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs e coli dna ligase
    Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and <t>FII.</t> The <t>DNA</t> substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.
    E Coli Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 468 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli dna ligase/product/New England Biolabs
    Average 99 stars, based on 468 article reviews
    Price from $9.99 to $1999.99
    e coli dna ligase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher e coli ligase
    Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand <t>cDNA</t> synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear <t>DNA</t> was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).
    E Coli Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 2064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli ligase/product/Thermo Fisher
    Average 96 stars, based on 2064 article reviews
    Price from $9.99 to $1999.99
    e coli ligase - by Bioz Stars, 2020-07
    96/100 stars
      Buy from Supplier

    95
    TaKaRa e coli dna ligase
    2.5% agarose gel electrophoreses for the three round <t>PCR</t> products. Electrophoreses were run in 1 x TAE at 60 V for 40 min. Lanes M and M2: <t>DNA</t> marker I and pUC19 DNA/MspI Marker, respectively; Lanes 1, 3, and 5: the first, second, and third round PCR products, respectively; Lanes 2, 4, and 6: the negative controls. ( A ) and ( B )The first round PCR templates were the ligation products of linkers A–B joined by T4 and E. coli DNA ligases, respectively. ( C ) and ( D ) The first round PCR templates were the ligation products of linkers C–D joined by T4 and E. coli DNA ligases, respectively. ( E ) The first round PCR templates were the ligation products cut from the denaturing PAGE gel.
    E Coli Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli dna ligase/product/TaKaRa
    Average 95 stars, based on 158 article reviews
    Price from $9.99 to $1999.99
    e coli dna ligase - by Bioz Stars, 2020-07
    95/100 stars
      Buy from Supplier

    92
    BIOKE e coli dna ligase
    2.5% agarose gel electrophoreses for the three round <t>PCR</t> products. Electrophoreses were run in 1 x TAE at 60 V for 40 min. Lanes M and M2: <t>DNA</t> marker I and pUC19 DNA/MspI Marker, respectively; Lanes 1, 3, and 5: the first, second, and third round PCR products, respectively; Lanes 2, 4, and 6: the negative controls. ( A ) and ( B )The first round PCR templates were the ligation products of linkers A–B joined by T4 and E. coli DNA ligases, respectively. ( C ) and ( D ) The first round PCR templates were the ligation products of linkers C–D joined by T4 and E. coli DNA ligases, respectively. ( E ) The first round PCR templates were the ligation products cut from the denaturing PAGE gel.
    E Coli Dna Ligase, supplied by BIOKE, 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/e coli dna ligase/product/BIOKE
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    e coli dna ligase - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.

    Journal: Molecular and Cellular Biology

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A

    doi: 10.1128/MCB.22.7.2037-2046.2002

    Figure Lengend Snippet: Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.

    Article Snippet: Like E. coli DNA ligase, FII could convert a nicked circular DNA substrate into a supercoiled molecule in the presence of ethidium bromide (Fig. ).

    Techniques: In Vitro, Fractionation, Incubation, Agarose Gel Electrophoresis, Staining

    Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

    Journal: Nucleic Acids Research

    Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae

    doi:

    Figure Lengend Snippet: Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

    Article Snippet: Total RNA (3 µg) was reverse transcribed and second strand cDNA synthesis was carried out with Escherichia coli DNA polymerase I, E.coli RNase H and E.coli DNA Ligase (SuperScript™ Choice System for cDNA Synthesis, Invitrogen).

    Techniques: Isolation, Agarose Gel Electrophoresis, Derivative Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Clone Assay

    RNAse protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 DNA used as a marker. The size of the bands in bp is indicated at both sides.

    Journal: Immunology

    Article Title: Structural and functional analysis of the human CD45 gene (PTPRC) upstream region: evidence for a functional promoter within the first intron of the gene

    doi: 10.1046/j.1365-2567.2001.01177.x

    Figure Lengend Snippet: RNAse protection assay. The upper panel shows a schematic representation of part of the 5′ region of the human CD45 gene and the RNA probes generated in vitro . The lower panel shows the protected fragments in different cell lines: Jurkat (human T cell), Raji (human B cell), K562 (human erythroid), U937 (human myeloid), EL-4 (mouse T cell) and HFB-1 (human B cell). M is the Hin fI-digested φX174 DNA used as a marker. The size of the bands in bp is indicated at both sides.

    Article Snippet: Second strand was generated by incubating the first-strand product with DNA polymerase I in the presence of RNase H and DNA ligase (all from Life Technologies) for 2 hr at 16°.

    Techniques: Rnase Protection Assay, Generated, In Vitro, Marker

    LPS enhances cGAS-mediated autophagy in A549 cells. Cells were either (A) pretreated with or without 400 ng/ml LPS for 4 h, followed by 2 µg/ml DNA transfection, (B) treated with the cGAS inhibitor RU at the indicated concentration and subsequently transfected with 2 µg/ml DNA or (C) pretreated with or without 400 ng/ml LPS for 4 h, followed by 2 µM RU treatment, and transfected with 2 µg/ml DNA. Following this cells were harvested 24-h post-transfection, and the protein expression of LC3-II and p62 was analyzed using western blot analysis. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Lipopolysaccharide enhances DNA-induced IFN-β expression and autophagy by upregulating cGAS expression in A549 cells

    doi: 10.3892/etm.2019.8001

    Figure Lengend Snippet: LPS enhances cGAS-mediated autophagy in A549 cells. Cells were either (A) pretreated with or without 400 ng/ml LPS for 4 h, followed by 2 µg/ml DNA transfection, (B) treated with the cGAS inhibitor RU at the indicated concentration and subsequently transfected with 2 µg/ml DNA or (C) pretreated with or without 400 ng/ml LPS for 4 h, followed by 2 µM RU treatment, and transfected with 2 µg/ml DNA. Following this cells were harvested 24-h post-transfection, and the protein expression of LC3-II and p62 was analyzed using western blot analysis. *P

    Article Snippet: For inhibitor, A549 cells were pretreated with TAK242 (10 µM), BX-795 (10 µM) or BAY11-7082 (20 µM) for 1 h, followed by LPS (400 ng/ml) treatment for 4 h. For transfection experiments, A549 cells (2×105 cells/well) were seeded in six-well plates overnight, then transfected with E. coli DNA (2 µg/ml) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

    Techniques: Transfection, Concentration Assay, Expressing, Western Blot

    2.5% agarose gel electrophoreses for the three round PCR products. Electrophoreses were run in 1 x TAE at 60 V for 40 min. Lanes M and M2: DNA marker I and pUC19 DNA/MspI Marker, respectively; Lanes 1, 3, and 5: the first, second, and third round PCR products, respectively; Lanes 2, 4, and 6: the negative controls. ( A ) and ( B )The first round PCR templates were the ligation products of linkers A–B joined by T4 and E. coli DNA ligases, respectively. ( C ) and ( D ) The first round PCR templates were the ligation products of linkers C–D joined by T4 and E. coli DNA ligases, respectively. ( E ) The first round PCR templates were the ligation products cut from the denaturing PAGE gel.

    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: 2.5% agarose gel electrophoreses for the three round PCR products. Electrophoreses were run in 1 x TAE at 60 V for 40 min. Lanes M and M2: DNA marker I and pUC19 DNA/MspI Marker, respectively; Lanes 1, 3, and 5: the first, second, and third round PCR products, respectively; Lanes 2, 4, and 6: the negative controls. ( A ) and ( B )The first round PCR templates were the ligation products of linkers A–B joined by T4 and E. coli DNA ligases, respectively. ( C ) and ( D ) The first round PCR templates were the ligation products of linkers C–D joined by T4 and E. coli DNA ligases, respectively. ( E ) The first round PCR templates were the ligation products cut from the denaturing PAGE gel.

    Article Snippet: Since the base deletion background of the ligation products generated by E. coli DNA ligase could also be significantly reduced by the purification of the ligation products with a PCR product purification kit, we speculated that the ligation products generated by E. coli DNA ligase might also contain the ligation products of the phosphorylated linkers, or in other words, perhaps the linkers with 5′-OH ends could be phosphorylated by E. coli DNA ligase, too.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Ligation, Polyacrylamide Gel Electrophoresis

    DNA sequencing results of the third round PCR products. The letters on the top are the expected DNA sequences. The downward arrows and the upward arrows indicate the ligation junctions of the sense strands and the antisense strands, respectively. ( A ) and ( B ) The sequencing templates were prepared from the ligation products of linkers A–B joined by T4 and E. coli DNA ligases, respectively. The ligation products were purified by using a PCR product purification kit before PCR. There was a 1-base deletion (-G) at the ligation junctions of both sense and antisense strands. The signal intensity from these deletions was only equivalent to about 25% of that from the normal sequences. ( C ) and ( D ) The sequencing templates were prepared from the ligation products of linkers C–D by T4 and E. coli DNA ligases, respectively. The ligation products were purified by using a PCR product purification kit before PCR. A 5-base deletion (-GGAGC) was found at the ligation junction of the antisense strand. The signal intensity from the deletion was only equivalent to about 25% of that from the normal sequence. ( E ) and ( F ) DNA sequencing template was prepared from the unpurified ligation products of linkers A–B and C–D, respectively. A 1-base deletion (-G) or a 5-base deletion (-GGAGC) was found at the ligation junctions of both sense and antisense strands of linkers A–B, or the ligation junction of the antisense strand of linkers C–D, respectively. The signal intensity from these deletions was equivalent to or even stronger than that from the normal sequence. ( G ) DNA sequencing template was prepared from the ligation products of linkers A–B cut from the denaturing PAGE gel. There was a 1-base deletion (-G) at the ligation junctions of both sense and antisense strands. ( H ) DNA sequencing template was prepared from the negative control of linkers A–B cut from the denaturing PAGE gel. There was 1-base deletion (-G) at the ligation junctions of both sense and antisense strands.

    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: DNA sequencing results of the third round PCR products. The letters on the top are the expected DNA sequences. The downward arrows and the upward arrows indicate the ligation junctions of the sense strands and the antisense strands, respectively. ( A ) and ( B ) The sequencing templates were prepared from the ligation products of linkers A–B joined by T4 and E. coli DNA ligases, respectively. The ligation products were purified by using a PCR product purification kit before PCR. There was a 1-base deletion (-G) at the ligation junctions of both sense and antisense strands. The signal intensity from these deletions was only equivalent to about 25% of that from the normal sequences. ( C ) and ( D ) The sequencing templates were prepared from the ligation products of linkers C–D by T4 and E. coli DNA ligases, respectively. The ligation products were purified by using a PCR product purification kit before PCR. A 5-base deletion (-GGAGC) was found at the ligation junction of the antisense strand. The signal intensity from the deletion was only equivalent to about 25% of that from the normal sequence. ( E ) and ( F ) DNA sequencing template was prepared from the unpurified ligation products of linkers A–B and C–D, respectively. A 1-base deletion (-G) or a 5-base deletion (-GGAGC) was found at the ligation junctions of both sense and antisense strands of linkers A–B, or the ligation junction of the antisense strand of linkers C–D, respectively. The signal intensity from these deletions was equivalent to or even stronger than that from the normal sequence. ( G ) DNA sequencing template was prepared from the ligation products of linkers A–B cut from the denaturing PAGE gel. There was a 1-base deletion (-G) at the ligation junctions of both sense and antisense strands. ( H ) DNA sequencing template was prepared from the negative control of linkers A–B cut from the denaturing PAGE gel. There was 1-base deletion (-G) at the ligation junctions of both sense and antisense strands.

    Article Snippet: Since the base deletion background of the ligation products generated by E. coli DNA ligase could also be significantly reduced by the purification of the ligation products with a PCR product purification kit, we speculated that the ligation products generated by E. coli DNA ligase might also contain the ligation products of the phosphorylated linkers, or in other words, perhaps the linkers with 5′-OH ends could be phosphorylated by E. coli DNA ligase, too.

    Techniques: DNA Sequencing, Polymerase Chain Reaction, Ligation, Sequencing, Purification, Polyacrylamide Gel Electrophoresis, Negative Control

    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: To see if T4 PNK could use NAD+ as its phosphate donor, oligos 2 and 3 of linkers A–B were separately preincubated with T4 PNK in 100 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligase buffer, 1 x BSA, 1 µM of each oligo, and 40 U of T4 PNK (Takara).

    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: To see if T4 PNK could use NAD+ as its phosphate donor, oligos 2 and 3 of linkers A–B were separately preincubated with T4 PNK in 100 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligase buffer, 1 x BSA, 1 µM of each oligo, and 40 U of T4 PNK (Takara).

    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: To see if T4 PNK could use NAD+ as its phosphate donor, oligos 2 and 3 of linkers A–B were separately preincubated with T4 PNK in 100 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligase buffer, 1 x BSA, 1 µM of each oligo, and 40 U of T4 PNK (Takara).

    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: To see if T4 PNK could use NAD+ as its phosphate donor, oligos 2 and 3 of linkers A–B were separately preincubated with T4 PNK in 100 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligase buffer, 1 x BSA, 1 µM of each oligo, and 40 U of T4 PNK (Takara).

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