t4 dna polymerase  (TaKaRa)

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

    TaKaRa t4 dna polymerase
    Absolute quantification of Tomato yellow leaf curl Sardinia virus (TYLCSV) virion-sense (VS) and complementary-sense (CS) ssDNA molecules, in the presence of an equimolar amount of dsDNA molecules of the same virus. Circular ssDNA molecules bearing the TYLCSV VS strand (10 6 copies) were mixed with the same quantity of circular ssDNA bearing the TYLCSV CS strand and with 10 6 molecules of phagemid dsDNA carrying the TYLCSV genome. The mix was denatured and used as template for <t>T4</t> DNA polymerase primer extension with the primers described in Fig. 1 , followed by qPCR with the indicated primer combinations for quantification of VS strands (grey bar), CS strands (white bar) and total viral DNA (VS and CS strands) (black bar). Data represent the average of three technical qPCR replicates.
    T4 DNA Polymerase catalyzes the 5 →3 incorporation of nucleotides into double stranded DNA using single stranded and primed DNA as a template It possesses strong 3 →5 exonuclease proofreading activity but does not exhibit 5 →3 exonuclease activity Typical T4 DNA polymerase protocols include 5 DNA overhang blunting as well as the generation of blunt double stranded DNA from double stranded DNA containing 3 overhangs These DNA products are often used in blunt cloning T4 DNA Polymerase is supplied in a buffer of 200 mM potassium phosphate pH 6 5 10 mM DTT and 50 glycerol
    https://www.bioz.com/result/t4 dna polymerase/product/TaKaRa
    Average 90 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-01
    90/100 stars

    Images

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep06438

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

    Techniques Used: Real-time Polymerase Chain Reaction

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

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

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

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

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

    doi: 10.1093/dnares/dsy018

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

    Techniques Used: Purification, DNA Purification

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

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

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

    doi: 10.1093/dnares/dsy018

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

    Techniques Used: Purification, DNA Purification

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni118

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl449

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl449

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

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

    Related Articles

    Clone Assay:

    Article Title: YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA
    Article Snippet: First, a 2 kb Hin dIII fragment (carrying a spectinomycin-resistance gene) of pHP45Ω ( ) was treated with T4 DNA polymerase and ligated with Dra I-digested pSTV28 (Takara Bio Inc.), yielding a pSTV28-derived vector with a spectinomycin-resistance gene instead of a chloramphenicol-resistance gene (named pSTD689). .. Then, a Sca I– Hin dIII ha-rseA fragment of pKK55 was cloned into pSTD689. pSTD714 (HA-RseA165) was constructed by cloning a PCR-amplified ha-rseA165 fragment into the Kpn I– Hin dIII sites of STD689.

    Article Title: Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2
    Article Snippet: To construct a plasmid for the benD gene disruption, a 9-kbp Bam HI fragment containing the benD gene was cloned by colony hybridization using a probe from a 2.8-kbp Bam HI- Pst I fragment of pBP144. .. The resultant pDBSC plasmid was digested with Sph I to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a Sma I ΩGmr cassette of pHP45Ωaac ( ).

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: For the construction of mutated GID1 s (including gid1-1:mGID1s ) in pBr BD-GID2L76A , previously constructed mutated GID1s ( ) were used as DNA templates to be amplified by PCR and cloned downstream of the MET25 promoter of pBr BD-GID2L76A to produce pBr BD-GID2L76A /3rd-mGID1s. .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: Hand2 Determines the Noradrenergic Phenotype in the Mouse Sympathetic Nervous System
    Article Snippet: .. The BsrDI end was polished with T4 DNA polymerase (TaKaRa) and cloned into the EcoRV site of PGKneoF2L2DTA. .. The 3′ fragment of Hand2 used to flank the 3′ loxP site was obtained from dH6.7pBKSII as a 2.7 kb SmaI fragment and was cloned into SmaI site of PGKneoF2L2DTA.

    Article Title: P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas
    Article Snippet: Paragraph title: Preparation of plasmid constructs and infectious clones. ... For P3N RB - PIPO CS , the cDNA fragment introduced into the pGEM-T Easy plasmid was cut with SacII, and the 5′ and 3′ ends of the plasmid were blunted by using T4 DNA polymerase (TaKaRa Bio, Kusatsu, Japan).

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

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

    Amplification:

    Article Title: Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses ▿Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses ▿ †
    Article Snippet: The fragmented DNA was polished using T4 DNA polymerase (Takara, Shiga, Japan) and T4 alkaline phosphatase supplied with deoxynucleoside triphosphate (dNTP) at 37°C for 30 min. .. The NotI linker (adapter) (long strand, 5′-AATTCGCGGCCGCGTCGAC-3′; short strand, 5′-phosphate-GTCGACGCGGCCGCG-3′) was ligated to the sheared DNA fragments using T4 DNA ligase for 1.5 h. The ligated DNA was PCR amplified with a primer (5′-CGGCCGCGTCGAC-3′) specific to the linker.

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: For the construction of mutated GID1 s (including gid1-1:mGID1s ) in pBr BD-GID2L76A , previously constructed mutated GID1s ( ) were used as DNA templates to be amplified by PCR and cloned downstream of the MET25 promoter of pBr BD-GID2L76A to produce pBr BD-GID2L76A /3rd-mGID1s. .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas
    Article Snippet: The P3N CS - PIPO RB fragment was amplified by using primer set 3946/3409 from the mixture of P3N of BY-CS and PIPO of Cl-RB amplified with primer sets 3946/3887 and 3954/3409, respectively. .. For P3N RB - PIPO CS , the cDNA fragment introduced into the pGEM-T Easy plasmid was cut with SacII, and the 5′ and 3′ ends of the plasmid were blunted by using T4 DNA polymerase (TaKaRa Bio, Kusatsu, Japan).

    Article Title: The Dominance of Alleles Controlling Self-Incompatibility in Brassica Pollen Is Regulated at the RNA Level
    Article Snippet: The insert DNA was amplified by PCR with LA Taq DNA polymerase (TaKaRa, Shiga, Japan) using specific primers corresponding to the vector (EMBL3F, 5′-CTTGCAGACAAACTGCGCAACTCGTGAAAG-3′; EMBL3R, 5′-GATCCACTCGTT-ATTCTCGGACGAGTGTTC-3′) and then fragmented using HydroShear (GeneMachines, San Carlos, CA). .. Fragments ranging from 1.5 to 3 kb were treated with T4 DNA polymerase (TaKaRa) and subcloned into pBluescript SK+ (Stratagene) for sequence analysis.

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

    Article Title: Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis
    Article Snippet: The upper and lower regions of ccpA were amplified by PCR with oligonucleotide primer pairs. .. Both of the PCR products were blunt ended by using T4 DNA polymerase (TaKaRa Shuzo).

    Filtration:

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring
    Article Snippet: The peak fractions were collected and further fractionated by gel filtration chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare) in R buffer containing 1 M NaCl. .. Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Synthesized:

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa). .. The chimeric double-stranded DNA thus obtained was transformed to E. coli strain JM109.

    Construct:

    Article Title: YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA
    Article Snippet: Plasmids encoding PDZ missense mutant forms of YaeL-His6 -Myc was constructed by introducing GGG (Gly214) to CAG (Gln), GCG (Ala234)/GCA (Ala235) to AAG (Lys)/AAG (Lys), GGC (Gly243) to CAG (Gln), GAC (Asp244) to AAG (Lys) and ATC (Ile246) to TAT (Tyr) codon alterations into yaeL-his 6 - myc on pKK11 by site-directed mutagenesis. pSTD691 (HA-RseA) was constructed as follows. .. First, a 2 kb Hin dIII fragment (carrying a spectinomycin-resistance gene) of pHP45Ω ( ) was treated with T4 DNA polymerase and ligated with Dra I-digested pSTV28 (Takara Bio Inc.), yielding a pSTV28-derived vector with a spectinomycin-resistance gene instead of a chloramphenicol-resistance gene (named pSTD689).

    Article Title: Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2
    Article Snippet: To construct a plasmid for the benD gene disruption, a 9-kbp Bam HI fragment containing the benD gene was cloned by colony hybridization using a probe from a 2.8-kbp Bam HI- Pst I fragment of pBP144. .. The resultant pDBSC plasmid was digested with Sph I to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a Sma I ΩGmr cassette of pHP45Ωaac ( ).

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: For the construction of mutated GID1 s (including gid1-1:mGID1s ) in pBr BD-GID2L76A , previously constructed mutated GID1s ( ) were used as DNA templates to be amplified by PCR and cloned downstream of the MET25 promoter of pBr BD-GID2L76A to produce pBr BD-GID2L76A /3rd-mGID1s. .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: Targeted Nuclear Import of Open Reading Frame 1 Protein Is Required for In Vivo Retrotransposition of a Telomere-Specific Non-Long Terminal Repeat Retrotransposon, SART1
    Article Snippet: Plasmids for the GFP expression experiment were constructed as follows. .. The Bgl II site of the plasmid was removed by Bgl II digestion and T4 DNA polymerase (TaKaRa) treatment in the presence of deoxynucleoside triphosphates (TaKaRa).

    Article Title: P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas
    Article Snippet: Paragraph title: Preparation of plasmid constructs and infectious clones. ... For P3N RB - PIPO CS , the cDNA fragment introduced into the pGEM-T Easy plasmid was cut with SacII, and the 5′ and 3′ ends of the plasmid were blunted by using T4 DNA polymerase (TaKaRa Bio, Kusatsu, Japan).

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: The reporter plasmid pnirKbgaH was constructed by the same procedure. .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa).

    Incubation:

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: The transformant was infected by an M13KO7 helper phage and then incubated in the kanamycin-supplemented medium. .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa).

    Article Title: A novel and simple method for construction of recombinant adenoviruses
    Article Snippet: Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR. .. The amplified fragments were incubated with 0.5 U of T4 DNA polymerase and 4 mM dGTP (TaKaRa) at 12°C for 45 min, as described previously ( , ).

    Introduce:

    Article Title: Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis
    Article Snippet: The ccpA-pi1 and ccpA-pi4 primers were designed to introduce BamHI and SalI restriction sites, respectively, at the 5′ end of each primer. .. Both of the PCR products were blunt ended by using T4 DNA polymerase (TaKaRa Shuzo).

    Expressing:

    Article Title: Targeted Nuclear Import of Open Reading Frame 1 Protein Is Required for In Vivo Retrotransposition of a Telomere-Specific Non-Long Terminal Repeat Retrotransposon, SART1
    Article Snippet: Plasmids for the GFP expression experiment were constructed as follows. .. The Bgl II site of the plasmid was removed by Bgl II digestion and T4 DNA polymerase (TaKaRa) treatment in the presence of deoxynucleoside triphosphates (TaKaRa).

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring
    Article Snippet: Paragraph title: Protein Expression and Purification ... Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Modification:

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: Site-directed mutagenesis of the promoter sequence of the nirK gene was carried out, according to Kunkel's method , with a slight modification. pUCpnirK, which contains the putative promoter sequence of the nirK gene in the pUC119 vector, was transformed to E. coli strain CJ236 ( dut1 ung1 thi-1 relA1 /pCJ105 [F′ Cmr ]). .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa).

    Reporter Assay:

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa). .. The mutant pUCpnirKG2C thus obtained was used for the preparation of plasmid pnirKG2C bgaH and transformant NKP03 (host strain, H. volcanii H26) for a reporter assay experiment.

    Transformation Assay:

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: The parental methylated and hemimethylated DNA in the PCR reaction mixture were digested with Dpn I, and the mutated SLR1 or GID2 cDNA that could not be digested with Dpn I was transformed into Escherichia coli XL10-Gold (Stratagene). .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: Site-directed mutagenesis of the promoter sequence of the nirK gene was carried out, according to Kunkel's method , with a slight modification. pUCpnirK, which contains the putative promoter sequence of the nirK gene in the pUC119 vector, was transformed to E. coli strain CJ236 ( dut1 ung1 thi-1 relA1 /pCJ105 [F′ Cmr ]). .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa).

    Hybridization:

    Article Title: Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2
    Article Snippet: To construct a plasmid for the benD gene disruption, a 9-kbp Bam HI fragment containing the benD gene was cloned by colony hybridization using a probe from a 2.8-kbp Bam HI- Pst I fragment of pBP144. .. The resultant pDBSC plasmid was digested with Sph I to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a Sma I ΩGmr cassette of pHP45Ωaac ( ).

    Electroporation:

    Article Title: Hand2 Determines the Noradrenergic Phenotype in the Mouse Sympathetic Nervous System
    Article Snippet: The BsrDI end was polished with T4 DNA polymerase (TaKaRa) and cloned into the EcoRV site of PGKneoF2L2DTA. .. Prior to ES cell electroporation, the targeting vector was linearized with XhoI.

    Inverse PCR:

    Article Title: Targeted Nuclear Import of Open Reading Frame 1 Protein Is Required for In Vivo Retrotransposition of a Telomere-Specific Non-Long Terminal Repeat Retrotransposon, SART1
    Article Snippet: The Bgl II site of the plasmid was removed by Bgl II digestion and T4 DNA polymerase (TaKaRa) treatment in the presence of deoxynucleoside triphosphates (TaKaRa). .. By using EGFPS814BglII and A813 primers, inverse PCR and self-ligation were performed, and the resulting plasmid was named pHEGFPB.

    Chromatography:

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring
    Article Snippet: The peak fractions were collected and further fractionated by gel filtration chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare) in R buffer containing 1 M NaCl. .. Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Ligation:

    Article Title: Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2
    Article Snippet: The resultant pDB90 plasmid was digested with Sac I, and the 8-kbp Sac I fragment was ligated by intramolecular ligation. .. The resultant pDBSC plasmid was digested with Sph I to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a Sma I ΩGmr cassette of pHP45Ωaac ( ).

    Infection:

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: The transformant was infected by an M13KO7 helper phage and then incubated in the kanamycin-supplemented medium. .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa).

    DNA Sequencing:

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa). .. The plasmid was isolated from the transformant, and the intended substitution was confirmed by DNA sequencing.

    Sequencing:

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: After sequencing, each mutated SLR1 or GID2 cDNA was digested with Eco RI- Sma I and cloned into pGADT7 or pBRIDGE containing full-length GID1 cDNA downstream of the MET25 promoter (pBr BD/ 3rd- GID1 , construction that can express GID1 protein as a third clone), respectively, to produce pGADT7-mSLR1s and pBr BD-mGID2s/3rd-GID1, respectively. pBr BD-GID2L76A was constructed by PCR amplifying GID2L76A with Eco RI and Sma I sites at 5′ and 3′ ends, respectively, and cloning the resulting fragment into the Eco RI- Sma I site of pBr vector. .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: The Dominance of Alleles Controlling Self-Incompatibility in Brassica Pollen Is Regulated at the RNA Level
    Article Snippet: .. Fragments ranging from 1.5 to 3 kb were treated with T4 DNA polymerase (TaKaRa) and subcloned into pBluescript SK+ (Stratagene) for sequence analysis. ..

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa). .. The chimeric double-stranded DNA thus obtained was transformed to E. coli strain JM109.

    Sonication:

    Article Title: Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses ▿Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses ▿ †
    Article Snippet: The DNA was sheared into small fragments by sonication for 3 s on ice to prevent heat denaturation ( ). .. The fragmented DNA was polished using T4 DNA polymerase (Takara, Shiga, Japan) and T4 alkaline phosphatase supplied with deoxynucleoside triphosphate (dNTP) at 37°C for 30 min.

    Recombinant:

    Article Title: Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis
    Article Snippet: Both of the PCR products were blunt ended by using T4 DNA polymerase (TaKaRa Shuzo). .. The recombinant plasmid was electroporated into a competent strain of S. bovis , strain 12U1, with a Genepulser (Bio-Rad) operated at 12.5 kV/cm and 200 Ω. Transformants were selected with 10 μg of erythromycin per ml.

    Methylation:

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: The parental methylated and hemimethylated DNA in the PCR reaction mixture were digested with Dpn I, and the mutated SLR1 or GID2 cDNA that could not be digested with Dpn I was transformed into Escherichia coli XL10-Gold (Stratagene). .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Mutagenesis:

    Article Title: YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA
    Article Snippet: Plasmids encoding PDZ missense mutant forms of YaeL-His6 -Myc was constructed by introducing GGG (Gly214) to CAG (Gln), GCG (Ala234)/GCA (Ala235) to AAG (Lys)/AAG (Lys), GGC (Gly243) to CAG (Gln), GAC (Asp244) to AAG (Lys) and ATC (Ile246) to TAT (Tyr) codon alterations into yaeL-his 6 - myc on pKK11 by site-directed mutagenesis. pSTD691 (HA-RseA) was constructed as follows. .. First, a 2 kb Hin dIII fragment (carrying a spectinomycin-resistance gene) of pHP45Ω ( ) was treated with T4 DNA polymerase and ligated with Dra I-digested pSTV28 (Takara Bio Inc.), yielding a pSTV28-derived vector with a spectinomycin-resistance gene instead of a chloramphenicol-resistance gene (named pSTD689).

    Article Title: Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2
    Article Snippet: The benD gene was disrupted by deleting most of the gene and using Ω interposon mutagenesis by the method of Schweizer ( ). .. The resultant pDBSC plasmid was digested with Sph I to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a Sma I ΩGmr cassette of pHP45Ωaac ( ).

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: Full-length SLR1 or GID2 cDNA was then PCR amplified using one set of mutagenized primers corresponding to each mutation. .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: .. After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa). .. The chimeric double-stranded DNA thus obtained was transformed to E. coli strain JM109.

    Article Title: Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis
    Article Snippet: Paragraph title: Construction of ccpA -disrupted mutant of S. bovis . ... Both of the PCR products were blunt ended by using T4 DNA polymerase (TaKaRa Shuzo).

    Isolation:

    Article Title: The Dominance of Alleles Controlling Self-Incompatibility in Brassica Pollen Is Regulated at the RNA Level
    Article Snippet: Fragments ranging from 1.5 to 3 kb were treated with T4 DNA polymerase (TaKaRa) and subcloned into pBluescript SK+ (Stratagene) for sequence analysis. .. One clone covering the ∼18.5-kb upstream region of SRK 60 was isolated and designated Bp120.

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

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa). .. The plasmid was isolated from the transformant, and the intended substitution was confirmed by DNA sequencing.

    Purification:

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

    Article Title: Hand2 Determines the Noradrenergic Phenotype in the Mouse Sympathetic Nervous System
    Article Snippet: The internal fragment that was placed between the first loxP site and the neomycin gene was obtained in two steps; a 6.7 kb SmaI-EcoRI fragment containing the 5′ untranslated region to 2 kb past the gene was purified from a P1 clone and cloned into SmaI-EcoRI the site of pBKSII (Stratagene) to generate dH6.7pBKSII. .. The BsrDI end was polished with T4 DNA polymerase (TaKaRa) and cloned into the EcoRV site of PGKneoF2L2DTA.

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring
    Article Snippet: Paragraph title: Protein Expression and Purification ... Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

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

    Polymerase Chain Reaction:

    Article Title: YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA
    Article Snippet: First, a 2 kb Hin dIII fragment (carrying a spectinomycin-resistance gene) of pHP45Ω ( ) was treated with T4 DNA polymerase and ligated with Dra I-digested pSTV28 (Takara Bio Inc.), yielding a pSTV28-derived vector with a spectinomycin-resistance gene instead of a chloramphenicol-resistance gene (named pSTD689). .. Then, a Sca I– Hin dIII ha-rseA fragment of pKK55 was cloned into pSTD689. pSTD714 (HA-RseA165) was constructed by cloning a PCR-amplified ha-rseA165 fragment into the Kpn I– Hin dIII sites of STD689.

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

    Article Title: Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses ▿Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses ▿ †
    Article Snippet: The fragmented DNA was polished using T4 DNA polymerase (Takara, Shiga, Japan) and T4 alkaline phosphatase supplied with deoxynucleoside triphosphate (dNTP) at 37°C for 30 min. .. The NotI linker (adapter) (long strand, 5′-AATTCGCGGCCGCGTCGAC-3′; short strand, 5′-phosphate-GTCGACGCGGCCGCG-3′) was ligated to the sheared DNA fragments using T4 DNA ligase for 1.5 h. The ligated DNA was PCR amplified with a primer (5′-CGGCCGCGTCGAC-3′) specific to the linker.

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: For the construction of mutated GID1 s (including gid1-1:mGID1s ) in pBr BD-GID2L76A , previously constructed mutated GID1s ( ) were used as DNA templates to be amplified by PCR and cloned downstream of the MET25 promoter of pBr BD-GID2L76A to produce pBr BD-GID2L76A /3rd-mGID1s. .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara).

    Article Title: The Dominance of Alleles Controlling Self-Incompatibility in Brassica Pollen Is Regulated at the RNA Level
    Article Snippet: The insert DNA was amplified by PCR with LA Taq DNA polymerase (TaKaRa, Shiga, Japan) using specific primers corresponding to the vector (EMBL3F, 5′-CTTGCAGACAAACTGCGCAACTCGTGAAAG-3′; EMBL3R, 5′-GATCCACTCGTT-ATTCTCGGACGAGTGTTC-3′) and then fragmented using HydroShear (GeneMachines, San Carlos, CA). .. Fragments ranging from 1.5 to 3 kb were treated with T4 DNA polymerase (TaKaRa) and subcloned into pBluescript SK+ (Stratagene) for sequence analysis.

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

    Article Title: Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis
    Article Snippet: .. Both of the PCR products were blunt ended by using T4 DNA polymerase (TaKaRa Shuzo). .. An erythromycin resistance gene, ermB , was inserted between the ccpA-pi1 - ccpA-pi2 fragment and the ccpA-pi3 - ccpA-pi4 fragment with T4 DNA ligase (TaKaRa Shuzo).

    Plasmid Preparation:

    Article Title: YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA
    Article Snippet: .. First, a 2 kb Hin dIII fragment (carrying a spectinomycin-resistance gene) of pHP45Ω ( ) was treated with T4 DNA polymerase and ligated with Dra I-digested pSTV28 (Takara Bio Inc.), yielding a pSTV28-derived vector with a spectinomycin-resistance gene instead of a chloramphenicol-resistance gene (named pSTD689). .. Then, a Sca I– Hin dIII ha-rseA fragment of pKK55 was cloned into pSTD689. pSTD714 (HA-RseA165) was constructed by cloning a PCR-amplified ha-rseA165 fragment into the Kpn I– Hin dIII sites of STD689.

    Article Title: Differential Expression of Two Catechol 1,2-Dioxygenases in Burkholderia sp. Strain TH2
    Article Snippet: .. The resultant pDBSC plasmid was digested with Sph I to remove the benD region, blunted with T4 DNA polymerase (Takara Shuzo Co.) and ligated with a Sma I ΩGmr cassette of pHP45Ωaac ( ). .. The resultant plasmid, pDBSCΩ, was digested with Eco RV.

    Article Title: Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C]Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice [C] [W]
    Article Snippet: .. In detail, mGID1s carrying a Sma I site at each end were digested with Sma I and ligated into pBr BD-GID2L76A plasmid digested with Bgl II and blunted with T4 DNA polymerase (Takara). ..

    Article Title: Hand2 Determines the Noradrenergic Phenotype in the Mouse Sympathetic Nervous System
    Article Snippet: Paragraph title: Construction of targeting vector ... The BsrDI end was polished with T4 DNA polymerase (TaKaRa) and cloned into the EcoRV site of PGKneoF2L2DTA.

    Article Title: Targeted Nuclear Import of Open Reading Frame 1 Protein Is Required for In Vivo Retrotransposition of a Telomere-Specific Non-Long Terminal Repeat Retrotransposon, SART1
    Article Snippet: .. The Bgl II site of the plasmid was removed by Bgl II digestion and T4 DNA polymerase (TaKaRa) treatment in the presence of deoxynucleoside triphosphates (TaKaRa). .. Next, to make a vector adapted for GFP fusion at the C terminus, we introduced a Bgl II site instead of the stop codon (TAA) of the GFP-coding region in the above plasmid.

    Article Title: P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas
    Article Snippet: .. For P3N RB - PIPO CS , the cDNA fragment introduced into the pGEM-T Easy plasmid was cut with SacII, and the 5′ and 3′ ends of the plasmid were blunted by using T4 DNA polymerase (TaKaRa Bio, Kusatsu, Japan). .. The blunted fragments were digested with XhoI and inserted into the WClMV vector cut with SmaI and XhoI.

    Article Title: The Dominance of Alleles Controlling Self-Incompatibility in Brassica Pollen Is Regulated at the RNA Level
    Article Snippet: The insert DNA was amplified by PCR with LA Taq DNA polymerase (TaKaRa, Shiga, Japan) using specific primers corresponding to the vector (EMBL3F, 5′-CTTGCAGACAAACTGCGCAACTCGTGAAAG-3′; EMBL3R, 5′-GATCCACTCGTT-ATTCTCGGACGAGTGTTC-3′) and then fragmented using HydroShear (GeneMachines, San Carlos, CA). .. Fragments ranging from 1.5 to 3 kb were treated with T4 DNA polymerase (TaKaRa) and subcloned into pBluescript SK+ (Stratagene) for sequence analysis.

    Article Title: Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO
    Article Snippet: Paragraph title: Reporter plasmid for narO , narA , and nirK gene promoters. ... After annealing with a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK was synthesized by using T4 DNA polymerase (TaKaRa).

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

    Article Title: Preliminary crystallographic analysis of the N--terminal domain of FILIA, a protein essential for embryogenesis
    Article Snippet: .. The linearized vector was processed with T4 DNA polymerase as described above, except that dGTP was substituted for dCTP. .. Finally, the vector was annealed with the processed PCR product at 295 K for 5 min, followed by another 5 min in the presence of 5 m M EDTA to boost the annealing efficiency.

    Article Title: Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis
    Article Snippet: Both of the PCR products were blunt ended by using T4 DNA polymerase (TaKaRa Shuzo). .. The ligated product was digested with BamHI and SalI and then introduced into plasmid pUC18.

    Agarose Gel Electrophoresis:

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase
    Article Snippet: After the DNase I digests were purified with a Qiaquick nucleotide removal kit (Qiagen), it was confirmed on a 2.5% Metaphore agarose gel (FMC, Rockland, Minn.) or a 2% agarose gel, the size of which was about 100 bp. .. The MURA reaction was performed with an automatic thermal cycler (Ericomp, San Diego, Calif.) for 30 cycles, with each cycle consisting of 94°C for 60 s, 54 to 62°C for 40 s, and 72°C for 60 s. The MURA products were purified with a Qiaquick PCR purification kit (Qiagen) and then treated with 1 U of T4 DNA polymerase (Takara) in 40 μl of the manufacturer's 1X buffer and 0.17 mM each dNTP for 10 min at 37°C or with 10 U of S1 nuclease (Takara) in 40 μl of the manufacturer's 1X buffer for 10 min at 25°C.

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

    Concentration Assay:

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring
    Article Snippet: The eluate was diluted with R buffer (20 mM Tris-HCl pH 7.5, 10% glycerol, 0.5 mM dithiothreitol) to a final NaCl concentration of 100 mM and loaded onto a 1 mL HiTrap Sepharose Q column (GE Healthcare). .. Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

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  • 90
    TaKaRa e coli 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: <t>DNA</t> 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.
    E Coli Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa 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: <t>DNA</t> 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 TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 dna ligase - by Bioz Stars, 2020-01
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    90
    TaKaRa t4 dna polymerase
    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: <t>DNA</t> 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 Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 41 article reviews
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    t4 dna polymerase - by Bioz Stars, 2020-01
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    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: Ligation Products of the Complementary Linkers To explore if the linkers with 5′-OH ends could be joined by commercial T4 and E. coli DNA ligase, linkers A–B, C–D, and E–F were joined by using commercial T4 or E. coli DNA ligase.

    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: Ligation Products of the Complementary Linkers To explore if the linkers with 5′-OH ends could be joined by commercial T4 and E. coli DNA ligase, linkers A–B, C–D, and E–F were joined by using commercial T4 or E. coli DNA ligase.

    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: Ligation Products of the Complementary Linkers To explore if the linkers with 5′-OH ends could be joined by commercial T4 and E. coli DNA ligase, linkers A–B, C–D, and E–F were joined by using commercial T4 or E. coli DNA ligase.

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

    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: Ligation Products of the Complementary Linkers To explore if the linkers with 5′-OH ends could be joined by commercial T4 and E. coli DNA ligase, linkers A–B, C–D, and E–F were joined by using commercial T4 or E. coli DNA ligase.

    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: Ligation Products of the Complementary Linkers To explore if the linkers with 5′-OH ends could be joined by commercial T4 and E. coli DNA ligase, linkers A–B, C–D, and E–F were joined by using commercial T4 or E. coli DNA ligase.

    Techniques: DNA Sequencing, Polymerase Chain Reaction, Ligation, Sequencing, Purification, Polyacrylamide Gel Electrophoresis, 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: Ligation Products of the Complementary Linkers To explore if the linkers with 5′-OH ends could be joined by commercial T4 and E. coli DNA ligase, linkers A–B, C–D, and E–F were joined by using commercial T4 or E. coli DNA ligase.

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