t4 dna polymerase  (New England Biolabs)


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    Name:
    T4 DNA Polymerase
    Description:

    Catalog Number:
    M0203L
    Price:
    None
    Score:
    85
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    Structured Review

    New England Biolabs t4 dna polymerase
    Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 16 article reviews
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    t4 dna polymerase - by Bioz Stars, 2019-12
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    Images

    1) Product Images from "Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes"

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020556

    Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.
    Figure Legend Snippet: Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    Techniques Used: Clone Assay, Activity Assay, Polymerase Chain Reaction, Sequencing, Incubation, Plasmid Preparation

    Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme  Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent  E. coli  DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).
    Figure Legend Snippet: Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

    Techniques Used: Amplification, Clone Assay, Purification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay

    Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C ,  D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.
    Figure Legend Snippet: Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C , D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Incubation, Agarose Gel Electrophoresis

    Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).
    Figure Legend Snippet: Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation, Generated, Agarose Gel Electrophoresis

    2) Product Images from "Directed evolution of protein enzymes using nonhomologous random recombination"

    Article Title: Directed evolution of protein enzymes using nonhomologous random recombination

    Journal:

    doi: 10.1073/pnas.0402202101

    Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry
    Figure Legend Snippet: Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry

    Techniques Used: Ligation

    3) Product Images from "Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases, Revealing their Potential to Lead to Genetic Change"

    Article Title: Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases, Revealing their Potential to Lead to Genetic Change

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080664

    Inhibition of Replicative and Translesion Polymerases by Intramolecular G-quadruplexes. In primer extension assays containing 75(3×GGG/*P31) and G-quadruplex-forming (4×GGG/*P31 or ext-4×GGG/*P31) substrates (0.2–0.3 nM) were incubated with increasing concentrations of the following polymerases: Kexo −  (10 and 20 U/L), T4 DNA polymerase (50 and 100 U/L), pol δ (3.4, 8.6, and 17.1 nM), human pol η (0.10 and 0.26 nM), pol κ (1.3 and 2.5 nM), pol μ (5.5, 10.9, and 27.3 nM), or pol β (11.6, 23.2, and 46.3 nM). Incubation temperatures and times for individual polymerases are specified above the gel. Markers (M) generated by 3×GGG/*P31 extension using dATP and dTTP only and Kexo −  (lanes 4, 15, 39, and 49) or T4 DNA polymerase (lanes 5, 16, 40, and 50) are denoted. Positions of partial extension products indicating polymerase stalling are denoted with brackets. White dashed lines indicate where separated groups of lanes from a single gel have been spliced together.
    Figure Legend Snippet: Inhibition of Replicative and Translesion Polymerases by Intramolecular G-quadruplexes. In primer extension assays containing 75(3×GGG/*P31) and G-quadruplex-forming (4×GGG/*P31 or ext-4×GGG/*P31) substrates (0.2–0.3 nM) were incubated with increasing concentrations of the following polymerases: Kexo − (10 and 20 U/L), T4 DNA polymerase (50 and 100 U/L), pol δ (3.4, 8.6, and 17.1 nM), human pol η (0.10 and 0.26 nM), pol κ (1.3 and 2.5 nM), pol μ (5.5, 10.9, and 27.3 nM), or pol β (11.6, 23.2, and 46.3 nM). Incubation temperatures and times for individual polymerases are specified above the gel. Markers (M) generated by 3×GGG/*P31 extension using dATP and dTTP only and Kexo − (lanes 4, 15, 39, and 49) or T4 DNA polymerase (lanes 5, 16, 40, and 50) are denoted. Positions of partial extension products indicating polymerase stalling are denoted with brackets. White dashed lines indicate where separated groups of lanes from a single gel have been spliced together.

    Techniques Used: Inhibition, Incubation, Generated

    4) Product Images from "One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies"

    Article Title: One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies

    Journal:

    doi: 10.1128/AEM.00844-12

    Optimization of one-step SLIC. (A) Effect of the duration of T4 DNA polymerase treatment on one-step SLIC. One hundred nanograms of BamHI-digested pUC118-HMG (4.9 kb) was mixed with an insert (Ton_0709, 1 kb, 22 bp of homology) at a 1:2 vector-to-insert
    Figure Legend Snippet: Optimization of one-step SLIC. (A) Effect of the duration of T4 DNA polymerase treatment on one-step SLIC. One hundred nanograms of BamHI-digested pUC118-HMG (4.9 kb) was mixed with an insert (Ton_0709, 1 kb, 22 bp of homology) at a 1:2 vector-to-insert

    Techniques Used: Plasmid Preparation

    5) Product Images from "Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis"

    Article Title: Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004682

    Effect of EXO1 on the structure of STN1-depleted telomeres. (A) TRF analysis; the asterisks indicate signal from interstitial telomeric DNA. (B) Quantification of the telomeric C-strand by dot-blot hybridization in G1  stn1  and G1  exo1 stn1  plants. Error bars show SDs from two independent samples; the P-value was calculated using a Student's t-test. (C) G-overhang analysis by the  in gel  hybridization technique. DNA samples pretreated with T4 DNA polymerase to remove 3′G-overhangs are indicated (3′ exo). The gels were first hybridized under nondenaturing conditions (top panels) and then denatured and hybridized again (bottom panels). (D) Quantification of G-overhang signals from a native gel. Error bars represent SDs from three (wt) and four ( stn1 ;  stn1 exo1 ) independent samples. (E) Frequency of telomeric sequence permutations forming the termini of blunt-ended telomeres. Error bars indicate SDs from five (wild-type) or four ( stn1 ,  exo stn1 ) biological replicates. Wild-type data are from   [31] .
    Figure Legend Snippet: Effect of EXO1 on the structure of STN1-depleted telomeres. (A) TRF analysis; the asterisks indicate signal from interstitial telomeric DNA. (B) Quantification of the telomeric C-strand by dot-blot hybridization in G1 stn1 and G1 exo1 stn1 plants. Error bars show SDs from two independent samples; the P-value was calculated using a Student's t-test. (C) G-overhang analysis by the in gel hybridization technique. DNA samples pretreated with T4 DNA polymerase to remove 3′G-overhangs are indicated (3′ exo). The gels were first hybridized under nondenaturing conditions (top panels) and then denatured and hybridized again (bottom panels). (D) Quantification of G-overhang signals from a native gel. Error bars represent SDs from three (wt) and four ( stn1 ; stn1 exo1 ) independent samples. (E) Frequency of telomeric sequence permutations forming the termini of blunt-ended telomeres. Error bars indicate SDs from five (wild-type) or four ( stn1 , exo stn1 ) biological replicates. Wild-type data are from [31] .

    Techniques Used: Dot Blot, Hybridization, Sequencing

    6) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in   Fig 2C  using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with  Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in   Fig 2E  lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either  Sca I or  Hin dIII and religated (lanes 4–7) or linearized with  Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and  Sca I-linearized DNA were ran in lanes 2–3 as migration markers.
    Figure Legend Snippet: Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in Fig 2C using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in Fig 2E lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either Sca I or Hin dIII and religated (lanes 4–7) or linearized with Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and Sca I-linearized DNA were ran in lanes 2–3 as migration markers.

    Techniques Used: Plasmid Preparation, Incubation, Marker, Negative Control, Migration, Isolation

    7) Product Images from "Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis"

    Article Title: Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004682

    Effect of EXO1 on the structure of STN1-depleted telomeres. (A) TRF analysis; the asterisks indicate signal from interstitial telomeric DNA. (B) Quantification of the telomeric C-strand by dot-blot hybridization in G1  stn1  and G1  exo1 stn1  plants. Error bars show SDs from two independent samples; the P-value was calculated using a Student's t-test. (C) G-overhang analysis by the  in gel  hybridization technique. DNA samples pretreated with T4 DNA polymerase to remove 3′G-overhangs are indicated (3′ exo). The gels were first hybridized under nondenaturing conditions (top panels) and then denatured and hybridized again (bottom panels). (D) Quantification of G-overhang signals from a native gel. Error bars represent SDs from three (wt) and four ( stn1 ;  stn1 exo1 ) independent samples. (E) Frequency of telomeric sequence permutations forming the termini of blunt-ended telomeres. Error bars indicate SDs from five (wild-type) or four ( stn1 ,  exo stn1 ) biological replicates. Wild-type data are from   [31] .
    Figure Legend Snippet: Effect of EXO1 on the structure of STN1-depleted telomeres. (A) TRF analysis; the asterisks indicate signal from interstitial telomeric DNA. (B) Quantification of the telomeric C-strand by dot-blot hybridization in G1 stn1 and G1 exo1 stn1 plants. Error bars show SDs from two independent samples; the P-value was calculated using a Student's t-test. (C) G-overhang analysis by the in gel hybridization technique. DNA samples pretreated with T4 DNA polymerase to remove 3′G-overhangs are indicated (3′ exo). The gels were first hybridized under nondenaturing conditions (top panels) and then denatured and hybridized again (bottom panels). (D) Quantification of G-overhang signals from a native gel. Error bars represent SDs from three (wt) and four ( stn1 ; stn1 exo1 ) independent samples. (E) Frequency of telomeric sequence permutations forming the termini of blunt-ended telomeres. Error bars indicate SDs from five (wild-type) or four ( stn1 , exo stn1 ) biological replicates. Wild-type data are from [31] .

    Techniques Used: Dot Blot, Hybridization, Sequencing

    8) Product Images from "Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection"

    Article Title: Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191315

    Diagrammatic representation of T7 promoter-lac operator-based pVMExp14367 expression vector to obtain recombinant proteins with N-terminal H10-TEV and C-terminal BAP tag. Only relevant genes and restriction sites are shown. The maps are not to scale. T7 lac , T7 promoter-lac operator; RBS, ribosome-binding site; H10, deca-histidine tag; TEV, Tobacco Etch Virus protease cleavage site; S, glycine-serine rich spacers; SacR-SacB, 2.0 kbp SacR-SacB gene cassette flanked by BsaI sites; BAP, Biotin Acceptor Peptide; T7Tn, T7 transcription terminator; f ori, origin of replication of filamentous phage; Amp r , beta-lactamase gene; ColE1 ori, origin of replication. The amino acids encoded are shown in single letter code (bold) above the nucleotide sequence (A-D). (A-C) The sequence of the important components of vector including 2 BsaI cloning sites. (D) Sequence flanking the gene of interest (GOI) after PCR amplification. (E) GOI carrying 5’- 4 base overhangs generated after treatment with T4 DNA polymerase in the presence of dTTP.
    Figure Legend Snippet: Diagrammatic representation of T7 promoter-lac operator-based pVMExp14367 expression vector to obtain recombinant proteins with N-terminal H10-TEV and C-terminal BAP tag. Only relevant genes and restriction sites are shown. The maps are not to scale. T7 lac , T7 promoter-lac operator; RBS, ribosome-binding site; H10, deca-histidine tag; TEV, Tobacco Etch Virus protease cleavage site; S, glycine-serine rich spacers; SacR-SacB, 2.0 kbp SacR-SacB gene cassette flanked by BsaI sites; BAP, Biotin Acceptor Peptide; T7Tn, T7 transcription terminator; f ori, origin of replication of filamentous phage; Amp r , beta-lactamase gene; ColE1 ori, origin of replication. The amino acids encoded are shown in single letter code (bold) above the nucleotide sequence (A-D). (A-C) The sequence of the important components of vector including 2 BsaI cloning sites. (D) Sequence flanking the gene of interest (GOI) after PCR amplification. (E) GOI carrying 5’- 4 base overhangs generated after treatment with T4 DNA polymerase in the presence of dTTP.

    Techniques Used: Expressing, Plasmid Preparation, Recombinant, Binding Assay, Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification, Generated

    9) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    Figure Legend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Techniques Used: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into  E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.
    Figure Legend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Techniques Used: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    10) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111538

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

    11) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111538

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

    12) Product Images from "An efficient method to assemble linear DNA templates for in vitro screening and selection systems"

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp589

    Efficiency of template assembly by uracil excision–ligation and the purifying PCR for DNA templates prepared with  Taq  DNA polymerase. Precipitation with PEG–MgCl 2  is necessary and sufficient for the efficient assembly. L1, hyperladder I in kilobase pairs; L2, substrate AGT amplified with  Taq , precipitated with PEG–MgCl 2  and blunted with T4 DNA polymerase; L3, assembly of L2 with its UTRs + Avi-tag; L4, purifying PCR of L3 with 50 ng DNA per 100 μl PCR; L5, purifying PCR of L3 with 250 ng DNA per 100 μl PCR; L6, purifying PCR of L3 with 500 ng DNA per 100 μl PCR; L7, hyperladder I in kb; L8, substrate AGT amplified with  Taq  and blunted with T4 DNA polymerase; L9, assembly of L8 with its UTRs + Avi-tag; L10, purifying PCR of L9 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.
    Figure Legend Snippet: Efficiency of template assembly by uracil excision–ligation and the purifying PCR for DNA templates prepared with Taq DNA polymerase. Precipitation with PEG–MgCl 2 is necessary and sufficient for the efficient assembly. L1, hyperladder I in kilobase pairs; L2, substrate AGT amplified with Taq , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L3, assembly of L2 with its UTRs + Avi-tag; L4, purifying PCR of L3 with 50 ng DNA per 100 μl PCR; L5, purifying PCR of L3 with 250 ng DNA per 100 μl PCR; L6, purifying PCR of L3 with 500 ng DNA per 100 μl PCR; L7, hyperladder I in kb; L8, substrate AGT amplified with Taq and blunted with T4 DNA polymerase; L9, assembly of L8 with its UTRs + Avi-tag; L10, purifying PCR of L9 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification

    ( A ) The enrichment of Avi-AGT DNA in model affinity selections relative to a non-binding, control template coding for AGT-FRB was  > 250-fold. L1, hyperladder I in kilobase pairs; L2, PCR amplification of the supernatant; L3, PCR amplification of the bead fraction. ( B ) Assembly of recovered DNA fragments; L4, hyperladder I in kilobase pairs; L5, substrate AGT recovered from the bead fraction with  PfuTurbo  C x , precipitated with PEG–MgCl 2  and blunted with T4 DNA polymerase; L6, assembly of L5 with its UTRs + Avi-tag. L7: purifying PCR of L6 with 50 ng DNA per 100 μl PCR; L8, hyperladder I in kilobase pairs; L9, substrate AGT recovered from the bead fraction with  PfuTurbo  C x  and blunted with T4 DNA polymerase; L10, assembly of L9 with its UTRs + Avi-tag; L11, purifying PCR of L10 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.
    Figure Legend Snippet: ( A ) The enrichment of Avi-AGT DNA in model affinity selections relative to a non-binding, control template coding for AGT-FRB was > 250-fold. L1, hyperladder I in kilobase pairs; L2, PCR amplification of the supernatant; L3, PCR amplification of the bead fraction. ( B ) Assembly of recovered DNA fragments; L4, hyperladder I in kilobase pairs; L5, substrate AGT recovered from the bead fraction with PfuTurbo C x , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L6, assembly of L5 with its UTRs + Avi-tag. L7: purifying PCR of L6 with 50 ng DNA per 100 μl PCR; L8, hyperladder I in kilobase pairs; L9, substrate AGT recovered from the bead fraction with PfuTurbo C x and blunted with T4 DNA polymerase; L10, assembly of L9 with its UTRs + Avi-tag; L11, purifying PCR of L10 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Amplification

    ( A ) Assembly Scheme. (i) GOI or a derivative library is amplified with primers that specifically incorporate uracil nucleotides close to both 5′-ends. (ii) Assembly of the GOI with its 5′- and 3′-untranslated regions including any constant protein-coding regions based on a coupled uracil excision–ligation strategy. (iii) Pure templates are obtained following a short-purifying PCR, which effectively ‘removes’ excess substrates and partially assembled intermediates. ( B ) Mechanism of coupled uracil excision–ligation: first, USER enzyme catalyses the excision of uracil from DNA, thereby leaving a single base pair gap and a 3′-extension provided the 5′-portion can dissociate. Complementary overlapping 3′-extensions then direct the assembly of DNA fragments which are covalently sealed by T4 DNA ligase.
    Figure Legend Snippet: ( A ) Assembly Scheme. (i) GOI or a derivative library is amplified with primers that specifically incorporate uracil nucleotides close to both 5′-ends. (ii) Assembly of the GOI with its 5′- and 3′-untranslated regions including any constant protein-coding regions based on a coupled uracil excision–ligation strategy. (iii) Pure templates are obtained following a short-purifying PCR, which effectively ‘removes’ excess substrates and partially assembled intermediates. ( B ) Mechanism of coupled uracil excision–ligation: first, USER enzyme catalyses the excision of uracil from DNA, thereby leaving a single base pair gap and a 3′-extension provided the 5′-portion can dissociate. Complementary overlapping 3′-extensions then direct the assembly of DNA fragments which are covalently sealed by T4 DNA ligase.

    Techniques Used: Amplification, Ligation, Polymerase Chain Reaction

    Assembly of an epPCR library. L1, hyperladder I in kilobase pairs;. L2, epPCR library prepared with the Genemorph II kit; L3, epPCR library of L2 re-amplified with  Taq  DNA polymerase and uracil-containing primers; L4, assembly of L3 with its UTRs+Avi-tag after it has been precipitated with PEG–MgCl 2  and blunted with T4 DNA polymerase; L5, purifying PCR of L4 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.
    Figure Legend Snippet: Assembly of an epPCR library. L1, hyperladder I in kilobase pairs;. L2, epPCR library prepared with the Genemorph II kit; L3, epPCR library of L2 re-amplified with Taq DNA polymerase and uracil-containing primers; L4, assembly of L3 with its UTRs+Avi-tag after it has been precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L5, purifying PCR of L4 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

    Techniques Used: Amplification, Polymerase Chain Reaction

    13) Product Images from "TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa) Seeds"

    Article Title: TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa) Seeds

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0143877

    Schematic representation of the modular vectors generated and the assembly of binary destination vectors. (A)  The modular vectors were designed for overexpression in plants, consisting of FS (flanking sequences), promoter, TALE, FokI and Ter (terminator). The expression of the TALEN cassettes of the modular vectors is driven by the promoters specified below. The CaMV35S promoter (double-enhancer version) and maize ubiquitin promoter were used for dicot or monocot plant transformation. A PacI endonuclease site was embedded into the FS used to obtain DNA fragments. The typical protein structure of an artificial TALEN composed of a repetitive DNA binding domain flanked by an N-terminal and a C-terminal was followed by the cleavage domain of FokI nuclease. The GoldyTALEN scaffold with N- and C-terminal truncations (N152/C63) fused with enhanced heterodimeric (ELD, KKR mutations) FokI domains is also provided. ( B ) A modified LIC method was implemented to enable high-throughput cloning of constructs for genome editing. The binary destination vector (pTALEN-DES) and DNA fragments containing TALEN cassettes were obtained using the restrictive endonuclease (RE) PacI and were processed by T4 DNA polymerase to create specific 12 nucleotide single-stranded overhangs. After annealing, a circular plasmid was generated in  Escherichia coli  cells without the use of T4 DNA ligase. ( C ) A schematic illustrating the construction of the binary destination vector containing four TALEN cassettes. RE, Restriction endonuclease; LB and RB, left border and right border of T-DNA; NptII, neomycin phosphotransferase (KanR); PacI, AgeI, SpeI, SbfI, recognition sequences of the restriction enzymes PacI, AgeI, SpeI and SbfI, respectively.
    Figure Legend Snippet: Schematic representation of the modular vectors generated and the assembly of binary destination vectors. (A) The modular vectors were designed for overexpression in plants, consisting of FS (flanking sequences), promoter, TALE, FokI and Ter (terminator). The expression of the TALEN cassettes of the modular vectors is driven by the promoters specified below. The CaMV35S promoter (double-enhancer version) and maize ubiquitin promoter were used for dicot or monocot plant transformation. A PacI endonuclease site was embedded into the FS used to obtain DNA fragments. The typical protein structure of an artificial TALEN composed of a repetitive DNA binding domain flanked by an N-terminal and a C-terminal was followed by the cleavage domain of FokI nuclease. The GoldyTALEN scaffold with N- and C-terminal truncations (N152/C63) fused with enhanced heterodimeric (ELD, KKR mutations) FokI domains is also provided. ( B ) A modified LIC method was implemented to enable high-throughput cloning of constructs for genome editing. The binary destination vector (pTALEN-DES) and DNA fragments containing TALEN cassettes were obtained using the restrictive endonuclease (RE) PacI and were processed by T4 DNA polymerase to create specific 12 nucleotide single-stranded overhangs. After annealing, a circular plasmid was generated in Escherichia coli cells without the use of T4 DNA ligase. ( C ) A schematic illustrating the construction of the binary destination vector containing four TALEN cassettes. RE, Restriction endonuclease; LB and RB, left border and right border of T-DNA; NptII, neomycin phosphotransferase (KanR); PacI, AgeI, SpeI, SbfI, recognition sequences of the restriction enzymes PacI, AgeI, SpeI and SbfI, respectively.

    Techniques Used: Generated, Over Expression, Expressing, Transformation Assay, Binding Assay, Modification, High Throughput Screening Assay, Clone Assay, Construct, Plasmid Preparation

    14) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111538

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

    15) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in   Fig 2C  using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with  Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in   Fig 2E  lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either  Sca I or  Hin dIII and religated (lanes 4–7) or linearized with  Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and  Sca I-linearized DNA were ran in lanes 2–3 as migration markers.
    Figure Legend Snippet: Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in Fig 2C using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in Fig 2E lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either Sca I or Hin dIII and religated (lanes 4–7) or linearized with Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and Sca I-linearized DNA were ran in lanes 2–3 as migration markers.

    Techniques Used: Plasmid Preparation, Incubation, Marker, Negative Control, Migration, Isolation

    16) Product Images from "Harnessing anthocyanin-rich fruit: a visible reporter for tracing virus-induced gene silencing in pepper fruit"

    Article Title: Harnessing anthocyanin-rich fruit: a visible reporter for tracing virus-induced gene silencing in pepper fruit

    Journal: Plant Methods

    doi: 10.1186/s13007-016-0151-5

    Cloning procedure using the  An2  reporter and the TRV2-LIC vector adapted from Dong et al. [  12 ]. The TRV2-LIC vector was digested with  Pst I and treated with T4 DNA polymerase and dTTP to generate sticky ends. The gene of interest was amplified by PCR using gene specific primers (GSP) and LIC (Gene_lic_F) and An2 adaptors (Gene_An2_R) using cDNA, and then treated with T4 DNA polymerase.  An2  was amplified using specific primers with an An2 adaptor (An2_F) and an LIC adaptor (An2_lic_R). The An2 adaptor sequence annealed to both the gene and  An2  fragments and a subsequent PCR using the LIC adaptor-attached primers fused the two fragments. The following PCR fragments were treated with T4 DNA polymerase and dATP to generate complementary sticky ends to anneal the ends of the linearized vector without DNA ligase. A mixture of both fragments was then transformed into  E. coli  DH5α
    Figure Legend Snippet: Cloning procedure using the An2 reporter and the TRV2-LIC vector adapted from Dong et al. [ 12 ]. The TRV2-LIC vector was digested with Pst I and treated with T4 DNA polymerase and dTTP to generate sticky ends. The gene of interest was amplified by PCR using gene specific primers (GSP) and LIC (Gene_lic_F) and An2 adaptors (Gene_An2_R) using cDNA, and then treated with T4 DNA polymerase. An2 was amplified using specific primers with an An2 adaptor (An2_F) and an LIC adaptor (An2_lic_R). The An2 adaptor sequence annealed to both the gene and An2 fragments and a subsequent PCR using the LIC adaptor-attached primers fused the two fragments. The following PCR fragments were treated with T4 DNA polymerase and dATP to generate complementary sticky ends to anneal the ends of the linearized vector without DNA ligase. A mixture of both fragments was then transformed into E. coli DH5α

    Techniques Used: Clone Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Sequencing, Transformation Assay

    17) Product Images from "Bleomycin-induced genome structural variations in normal, non-tumor cells"

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34580-8

    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.
    Figure Legend Snippet: Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.

    Techniques Used: Ligation, Sequencing, Hybridization, Two Tailed Test

    18) Product Images from "Nucleic acid evolution and minimization by nonhomologous random recombination"

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    Journal: Nature biotechnology

    doi: 10.1038/nbt736

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Figure Legend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Techniques Used: Ligation, Binding Assay

    19) Product Images from "Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes"

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020556

    Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.
    Figure Legend Snippet: Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    Techniques Used: Clone Assay, Activity Assay, Polymerase Chain Reaction, Sequencing, Incubation, Plasmid Preparation

    Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme  Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent  E. coli  DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).
    Figure Legend Snippet: Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

    Techniques Used: Amplification, Clone Assay, Purification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay

    Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C ,  D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.
    Figure Legend Snippet: Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C , D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Incubation, Agarose Gel Electrophoresis

    Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).
    Figure Legend Snippet: Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation, Generated, Agarose Gel Electrophoresis

    20) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111538

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

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    Clone Assay:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: The cross-links were reversed, and the samples were sequentially treated with RNase A and proteinase K, and then isolated as performed in the ChIP assay. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector. .. To determine the location and flanking genes on the RNF2-responding loci on the chromosomes, a Human BLAST Search of UCSC Genome Bioinformatics ( ) and the BLAST of NCBI ( ) was used.

    Article Title: A mutation in the catalytic subunit of yeast telomerase alters primer-template alignment while promoting processivity and protein-DNA binding
    Article Snippet: Paragraph title: Telomere DNA cloning and sequence analysis ... RNase-treated DNA samples (~20–40 μg) were blunted by treatment with 4.5 U of T4 DNA polymerase (NEB) in the presence of 1 mM dNTPs at 12°C.

    Article Title: Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap
    Article Snippet: A 1,962 bp PmeI/BsrBI restriction fragment was isolated from pKW2 and cloned into pBB5 (a derivative of pBB4 that has an E. coli backbone modification), at the EcoRV site located in exon 9 of the GGTA1 locus, to build pBB6. .. To isolate the CAG promoter, pCAG-Cre:GFP (Addgene plasmid 13776; [ ]) was cut with EcoRI and SalI, and the 3` ends were extended with T4 DNA polymerase (New England Biolabs), to produce blunt ends.

    Article Title: Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguini
    Article Snippet: We began with plasmid pJFP96, which contains 1.35 kb of S. sanguinis DNA encompassing the SSA_0169 gene and flanking sequences, with the SSA_0169 gene interrupted by the aad9 gene encoding resistance to Spc.4 We first deleted HindIII and SphI restriction sites to facilitate downstream cloning reactions. .. The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: Paragraph title: 2.3.9. Ligation independent cloning (LIC) ... An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product.

    Article Title: Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites
    Article Snippet: pLF34 was provided by David Kaback (Dept. of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, N.J.). .. The 1.5-kb Kpn I- Bgl II fragment of pLF34 was cloned into pBS-KS (Stratagene); the 3.0-kb Fsp I fragment of this construct was ligated to the 4.7-kb Fsp I fragment of pRS326 ( ) and the 2.6-kb Fsp I fragment of pRS306 ( ) to yield yAR8KG and yAR8KG0, respectively. yAR8KG was isolated from E. coli strain GM2929; the 650-bp Eco RV- Cla I fragment was blunted with T4 polymerase (New England Biolabs [NEB]) plus deoxynucleoside triphosphates (dNTPs) (Roche Molecular Biochemicals), Hin dIII linkers (NEB) were attached, and the fragment was cloned into pRS326 to yield yAR8VCA, which contains the Eco RV- Cla I fragment in the same orientation as in yAR8KG. .. The Eco RV- Cla I fragment contains nucleotides 159457 to 160108 of the chromosome I sequence.

    Centrifugation:

    Article Title: A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
    Article Snippet: For T4 treatment (New England Biolabs), 200 to 400 ng of linearized vector, 4 μL 10× T4 buffer, 4 μL 100 m m dCTP, 2 μL 100 m m dithiothreitol, 0.4 μL bovine serum albumin, 0.8 μL T4 DNA polymerase (New England Biolabs), and water to 40 μL total volume are mixed. .. For T4 treatment (New England Biolabs), 200 to 400 ng of linearized vector, 4 μL 10× T4 buffer, 4 μL 100 m m dCTP, 2 μL 100 m m dithiothreitol, 0.4 μL bovine serum albumin, 0.8 μL T4 DNA polymerase (New England Biolabs), and water to 40 μL total volume are mixed.

    Amplification:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: To detect the site occupied by the RNF2 protein on the human chromatin, the immunoprecipitated DNA fragments were amplified using following primer pairs: F21-17, 5′-ACA CAG TGA GAC TCT GTC TC-3’ and 5′-CGT GTT ACC ATG TCT GTC TAA A-3′; G16-18, 5′-AAG AGG TGC AAC CCA AC-3′ and 5′-GGC AAA ACC CTG TCT CTA CT-3′; F2-23, 5′-TTC TTT CAC TTA AAA GAA AGA CAC-3′ and 5’-TCT AAT ATT AGG GGT GGG AAA-3′; G11-18, 5′-TGG GCC AAG TCC AC-3′ and 5′-TCT CAT CCA AGT CAG GAC A-3′; β-actin, 5′-GCA GGC CTG GCC ATG CG-3′ and 5′-AGT TTT GGC GTT GGC CGC CTT-3′. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Article Title: A mutation in the catalytic subunit of yeast telomerase alters primer-template alignment while promoting processivity and protein-DNA binding
    Article Snippet: RNase-treated DNA samples (~20–40 μg) were blunted by treatment with 4.5 U of T4 DNA polymerase (NEB) in the presence of 1 mM dNTPs at 12°C. .. The samples were ethanol precipitated and 40 ng of a double-stranded oligonucleotide (created by annealing primers ds oligo 1 and ds oligo 2; Table S2) was incubated with the genomic DNA and 20 U of T4 DNA ligase (NEB) overnight at 17°C.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: In the current study, the primers for the second LRP amplification were replaced with WF5T4P (5′-ctc acc tta att aac ata gtg gtc tgc gga acc ggt -3′) and WR55T4P (5′-ata cct ccc ttc tac aat ggc agc cct gcc tcc tct gg -3′). .. An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product.

    DNA Ligation:

    Article Title:
    Article Snippet: To generate the blank control vector of p300-HA (CMVβ(Δp300)), the p300-HA plasmid was digested with NotI and HindIII. .. The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The FLAG-PCAF (p300/CBP-associated factor) expression vector was kindly provided by Dr. Yoshihiro Nakatani (Dana Farber Cancer Institute, Boston).

    Synthesized:

    Article Title: Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap
    Article Snippet: The loxP-IRES-mNeoR-loxP was isolated from a chemically synthesized in-house plasmid (pKW2), that contained all four components. .. To isolate the CAG promoter, pCAG-Cre:GFP (Addgene plasmid 13776; [ ]) was cut with EcoRI and SalI, and the 3` ends were extended with T4 DNA polymerase (New England Biolabs), to produce blunt ends.

    Article Title: 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation
    Article Snippet: The synthetic sequences were synthesized and subcloned into pIDTSMART plasmids by Integrated DNA Technologies (IDT). .. To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S).

    Construct:

    Article Title:
    Article Snippet: Paragraph title: Constructs and Transfection ... The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio).

    Article Title: Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap
    Article Snippet: Paragraph title: GGTA 1 targeting constructs ... To isolate the CAG promoter, pCAG-Cre:GFP (Addgene plasmid 13776; [ ]) was cut with EcoRI and SalI, and the 3` ends were extended with T4 DNA polymerase (New England Biolabs), to produce blunt ends.

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: Subsequently, the primers were used to construct the mutants by conventional PCR. .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Article Title: Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguini
    Article Snippet: The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106. .. The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106.

    Article Title: Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites
    Article Snippet: pLF34 was provided by David Kaback (Dept. of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, N.J.). .. The 1.5-kb Kpn I- Bgl II fragment of pLF34 was cloned into pBS-KS (Stratagene); the 3.0-kb Fsp I fragment of this construct was ligated to the 4.7-kb Fsp I fragment of pRS326 ( ) and the 2.6-kb Fsp I fragment of pRS306 ( ) to yield yAR8KG and yAR8KG0, respectively. yAR8KG was isolated from E. coli strain GM2929; the 650-bp Eco RV- Cla I fragment was blunted with T4 polymerase (New England Biolabs [NEB]) plus deoxynucleoside triphosphates (dNTPs) (Roche Molecular Biochemicals), Hin dIII linkers (NEB) were attached, and the fragment was cloned into pRS326 to yield yAR8VCA, which contains the Eco RV- Cla I fragment in the same orientation as in yAR8KG. .. The Eco RV- Cla I fragment contains nucleotides 159457 to 160108 of the chromosome I sequence.

    Incubation:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: Once again, the same antibodies as were used in the first immunoprecipitation were added into the diluted eluants and incubation, bead binding, washing and elution steps were sequentially repeated. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: In the current study, the primers for the second LRP amplification were replaced with WF5T4P (5′-ctc acc tta att aac ata gtg gtc tgc gga acc ggt -3′) and WR55T4P (5′-ata cct ccc ttc tac aat ggc agc cct gcc tcc tct gg -3′). .. An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product. .. Similarly, pCloneK was amplified fully with primers OriFT4P (5′-tat gtt aat taa ggt gag cgg tgt gaa ata ccg cac ag -3) and OriRT4P (5′-att gta gaa ggg agg tat ccg att tcg gcc tat tgg tt -3′).

    Luciferase:

    Article Title:
    Article Snippet: The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The GATA4-FLAG expression vector was kindly provided by Dr. Mona Nemer (University of Ottawa, Ottawa, Canada).

    Activity Assay:

    Article Title: Poly(A) Binding Protein C1 Is Essential for Efficient L1 Retrotransposition and Affects L1 RNP Formation
    Article Snippet: As illustrated in , a unique BstZ17I restriction site is located in the middle of this adaptor fragment, containing two “C-less” 18-bp sequences (blue and green). .. These two sequences can be removed upon BstZ17I digestion and the action of the (3′-to-5′) exonuclease activity of T4 DNA polymerase in the presence of dCTP. .. Similarly, two 18-bp overhangs can be produced on the PCR insert in the presence of T4 DNA polymerase and dGTP.

    Expressing:

    Article Title:
    Article Snippet: FLAG-p300 and p300-HA expression constructs were kindly provided by Dr. Richard Eckner (University of Zurich, Zurich, Switzerland) and Dr. Masaaki Ikeda (Tokyo Medical and Dental University, Tokyo, Japan). .. The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio).

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: The RGD deletion mutants were produced by digesting the pCDNA3-IL-32β/IL-32γ expression plasmids with HindIII (New England Biolabs, Ipswich, MA) and XbaI (New England Biolabs) to separate the backbone and insert. .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Modification:

    Article Title: Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap
    Article Snippet: A 1,962 bp PmeI/BsrBI restriction fragment was isolated from pKW2 and cloned into pBB5 (a derivative of pBB4 that has an E. coli backbone modification), at the EcoRV site located in exon 9 of the GGTA1 locus, to build pBB6. .. To isolate the CAG promoter, pCAG-Cre:GFP (Addgene plasmid 13776; [ ]) was cut with EcoRI and SalI, and the 3` ends were extended with T4 DNA polymerase (New England Biolabs), to produce blunt ends.

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: Subsequently, the modified IL-32β/IL-32γ inserts were ligated at the HindIII overhang into the previously isolated backbone, which also contained a HindIII overhang, by using T4 DNA ligase (New England Biolabs). .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Transformation Assay:

    Article Title: Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguini
    Article Snippet: Transformation of S. sanguinis with each construct was carried out as described previously ( ) except that cells were grown anaerobically prior to and after transformation. .. The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106.

    Article Title: Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans
    Article Snippet: Restriction endonucleases, T4 DNA ligase, alkaline phosphatase, and the Klenow fragment of DNA polymerase I were purchased from New England Biolabs (Beverly, Mass.) and used as recommended by the manufacturer. .. Plasmid DNA was extracted by using a QIAprep plasmid kit (QIAGEN).

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers. .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Countercurrent Chromatography:

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: In the current study, the primers for the second LRP amplification were replaced with WF5T4P (5′-ctc acc tta att aac ata gtg gtc tgc gga acc ggt -3′) and WR55T4P (5′-ata cct ccc ttc tac aat ggc agc cct gcc tcc tct gg -3′). .. An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product.

    Electroporation:

    Article Title: Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans
    Article Snippet: Restriction endonucleases, T4 DNA ligase, alkaline phosphatase, and the Klenow fragment of DNA polymerase I were purchased from New England Biolabs (Beverly, Mass.) and used as recommended by the manufacturer. .. Restriction endonucleases, T4 DNA ligase, alkaline phosphatase, and the Klenow fragment of DNA polymerase I were purchased from New England Biolabs (Beverly, Mass.) and used as recommended by the manufacturer.

    Transfection:

    Article Title:
    Article Snippet: Paragraph title: Constructs and Transfection ... The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio).

    Ligation:

    Article Title: A mutation in the catalytic subunit of yeast telomerase alters primer-template alignment while promoting processivity and protein-DNA binding
    Article Snippet: RNase-treated DNA samples (~20–40 μg) were blunted by treatment with 4.5 U of T4 DNA polymerase (NEB) in the presence of 1 mM dNTPs at 12°C. .. The samples were ethanol precipitated and 40 ng of a double-stranded oligonucleotide (created by annealing primers ds oligo 1 and ds oligo 2; Table S2) was incubated with the genomic DNA and 20 U of T4 DNA ligase (NEB) overnight at 17°C.

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: Subsequently, the modified IL-32β/IL-32γ inserts were ligated at the HindIII overhang into the previously isolated backbone, which also contained a HindIII overhang, by using T4 DNA ligase (New England Biolabs). .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs). .. Exactly after the arginine amino acid, a stop codon was present because of the restriction and ligation reaction.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: Paragraph title: 2.3.9. Ligation independent cloning (LIC) ... An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product.

    Protease Inhibitor:

    Article Title: ChIP-exo: A Method to Identify Genomic Location of DNA-binding proteins at Near Single Nucleotide Accuracy
    Article Snippet: Yeast culture .. 37% formaldehyde 2.5 M glycine ST buffer (see recipe) Protease inhibitor cocktail tablets (Roche) FA-lysis buffer (see recipe) 20% SDS (volume/volume) Antibody against the protein of interest Protein A- or G- Sepharose beads or equivalent (e.g. magnetic beads) FA-high salt wash buffer (see recipe) FA-wash 2 buffer (see recipe) FA-wash 3 buffer (see recipe) TE buffer (see recipe) 10 mM Tris-HCl (pH 8.0) NEBuffer 2 (New England BioLabs) 10× BSA (1mg/ml) 3 mM dNTPs T4 DNA polymerase (3 U/ul, New England BioLabs) 10 mM Tris-HCl (pH 7.5) 10× T4 DNA ligase buffer (New England BioLabs) T4 Polynucleotide Kinase (10 U/ul, New England BioLabs) 3 mM dATP Klenow Fragment (3′→5′ exo-) (5 U/ul, New England BioLabs) T4 DNA ligase (500 U/ul, New England BioLabs) 10× phi29 DNA polymerase buffer (New England BioLabs) phi29 DNA polymerase (10 U/ul, New England BioLabs) 10 mM Tris-HCl (pH 9.2) 10× lambda exonuclease buffer (New England BioLabs) Lambda exonulease (5 U/ul, New England BioLabs) RecJf exonuclease (30 U/ul, New England BioLabs) ChIP elution buffer (see recipe) Protease K (20 ug/ul, Roche) 25:24:1 phenol/chloroform/isoamyl alcohol (Sigma) 100% ethanol (Any supplier) 75% (volume/volume) ethanol Glycogen (20 mg/ml, Roche) AMPure magnetic beads (Agencourt) 25 mM dNTPs 10× standard taq reaction buffer (New England BioLabs) Taq DNA polymerase (5 U/ul, New England BioLabs) .. P1-T Adaptor, 15 uM (Life Technologies) 5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′ (41 bp) 5′-TCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGCC-3′ (42 bp) P2-T Adaptor, 15 uM (Life Technologies) 5′-CGCCTTGGCCGTACAGCAGCCTCTTACACAGAGAATGAGGAACCCGGGGCAGTT-3′ (55 bp) 5′-CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT-3′ (53 bp) Library PCR Primer 1, 20 uM (Life Technologies) 5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′ (28 bp) Library PCR Pimer 2, 20 uM (Life Technologies) 5′-CTGCCCCGGGTTCCTCATTCT-3′ (21 bp) Oligonucleotide sequences © Copyright 2008.

    Generated:

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: In the current study, the primers for the second LRP amplification were replaced with WF5T4P (5′-ctc acc tta att aac ata gtg gtc tgc gga acc ggt -3′) and WR55T4P (5′-ata cct ccc ttc tac aat ggc agc cct gcc tcc tct gg -3′). .. An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product. .. Similarly, pCloneK was amplified fully with primers OriFT4P (5′-tat gtt aat taa ggt gag cgg tgt gaa ata ccg cac ag -3) and OriRT4P (5′-att gta gaa ggg agg tat ccg att tcg gcc tat tgg tt -3′).

    other:

    Article Title: Protein Switch Engineering by Domain Insertion
    Article Snippet: Repair the digested DNA (to create blunt ends) using T4 DNA ligase and T4 DNA polymerase (New England Biolabs, Ipswich, MA).

    Article Title: Identification of a Novel Bacteriocin Regulatory System in Streptococcus mutans
    Article Snippet: Phusion DNA polymerase, restriction enzymes, T4 DNA ligase, and other DNA modifying enzymes were all purchased from New England Biolabs.

    DNA Sequencing:

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Nucleotide sequencing of both strands of purified PCR products was done at the DNA Sequencing Service of Sistemas Genómicos (Valencia, Spain). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Isolation:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: The cross-links were reversed, and the samples were sequentially treated with RNase A and proteinase K, and then isolated as performed in the ChIP assay. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector. .. To determine the location and flanking genes on the RNF2-responding loci on the chromosomes, a Human BLAST Search of UCSC Genome Bioinformatics ( ) and the BLAST of NCBI ( ) was used.

    Article Title: Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap
    Article Snippet: The hCD55, lambda attB, and the SV40 poly (A) components were isolated from cDNA clone MGC: 5192 IMAGE: 3460621 (Open Biosystems #3460621). .. To isolate the CAG promoter, pCAG-Cre:GFP (Addgene plasmid 13776; [ ]) was cut with EcoRI and SalI, and the 3` ends were extended with T4 DNA polymerase (New England Biolabs), to produce blunt ends.

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: Subsequently, the modified IL-32β/IL-32γ inserts were ligated at the HindIII overhang into the previously isolated backbone, which also contained a HindIII overhang, by using T4 DNA ligase (New England Biolabs). .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Article Title: 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation
    Article Snippet: To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S). .. To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S).

    Article Title: Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites
    Article Snippet: pLF34 was provided by David Kaback (Dept. of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, N.J.). .. The 1.5-kb Kpn I- Bgl II fragment of pLF34 was cloned into pBS-KS (Stratagene); the 3.0-kb Fsp I fragment of this construct was ligated to the 4.7-kb Fsp I fragment of pRS326 ( ) and the 2.6-kb Fsp I fragment of pRS306 ( ) to yield yAR8KG and yAR8KG0, respectively. yAR8KG was isolated from E. coli strain GM2929; the 650-bp Eco RV- Cla I fragment was blunted with T4 polymerase (New England Biolabs [NEB]) plus deoxynucleoside triphosphates (dNTPs) (Roche Molecular Biochemicals), Hin dIII linkers (NEB) were attached, and the fragment was cloned into pRS326 to yield yAR8VCA, which contains the Eco RV- Cla I fragment in the same orientation as in yAR8KG. .. The Eco RV- Cla I fragment contains nucleotides 159457 to 160108 of the chromosome I sequence.

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Total genomic DNA from recombinant yeasts was isolated using the Wizard DNA Purification kit (Promega Corporation, Madison, WI). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Sequencing:

    Article Title: A mutation in the catalytic subunit of yeast telomerase alters primer-template alignment while promoting processivity and protein-DNA binding
    Article Snippet: Paragraph title: Telomere DNA cloning and sequence analysis ... RNase-treated DNA samples (~20–40 μg) were blunted by treatment with 4.5 U of T4 DNA polymerase (NEB) in the presence of 1 mM dNTPs at 12°C.

    Article Title:
    Article Snippet: The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The GATA4-FLAG expression vector was kindly provided by Dr. Mona Nemer (University of Ottawa, Ottawa, Canada).

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: The inserts were digested with BtgI (New England Biolabs) to remove the GD amino acid sequence until the stop codon. .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Article Title: 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation
    Article Snippet: To design the synthetic DNA fragments, we utilized the wild-type Alu Ya5 sequence or the wild-type Alu Jb sequence as templates. .. To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S).

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Nucleotide sequencing of both strands of purified PCR products was done at the DNA Sequencing Service of Sistemas Genómicos (Valencia, Spain). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Binding Assay:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: Once again, the same antibodies as were used in the first immunoprecipitation were added into the diluted eluants and incubation, bead binding, washing and elution steps were sequentially repeated. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    DNA Extraction:

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Paragraph title: DNA isolation and manipulation. ... Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Magnetic Beads:

    Article Title: ChIP-exo: A Method to Identify Genomic Location of DNA-binding proteins at Near Single Nucleotide Accuracy
    Article Snippet: Yeast culture .. 37% formaldehyde 2.5 M glycine ST buffer (see recipe) Protease inhibitor cocktail tablets (Roche) FA-lysis buffer (see recipe) 20% SDS (volume/volume) Antibody against the protein of interest Protein A- or G- Sepharose beads or equivalent (e.g. magnetic beads) FA-high salt wash buffer (see recipe) FA-wash 2 buffer (see recipe) FA-wash 3 buffer (see recipe) TE buffer (see recipe) 10 mM Tris-HCl (pH 8.0) NEBuffer 2 (New England BioLabs) 10× BSA (1mg/ml) 3 mM dNTPs T4 DNA polymerase (3 U/ul, New England BioLabs) 10 mM Tris-HCl (pH 7.5) 10× T4 DNA ligase buffer (New England BioLabs) T4 Polynucleotide Kinase (10 U/ul, New England BioLabs) 3 mM dATP Klenow Fragment (3′→5′ exo-) (5 U/ul, New England BioLabs) T4 DNA ligase (500 U/ul, New England BioLabs) 10× phi29 DNA polymerase buffer (New England BioLabs) phi29 DNA polymerase (10 U/ul, New England BioLabs) 10 mM Tris-HCl (pH 9.2) 10× lambda exonuclease buffer (New England BioLabs) Lambda exonulease (5 U/ul, New England BioLabs) RecJf exonuclease (30 U/ul, New England BioLabs) ChIP elution buffer (see recipe) Protease K (20 ug/ul, Roche) 25:24:1 phenol/chloroform/isoamyl alcohol (Sigma) 100% ethanol (Any supplier) 75% (volume/volume) ethanol Glycogen (20 mg/ml, Roche) AMPure magnetic beads (Agencourt) 25 mM dNTPs 10× standard taq reaction buffer (New England BioLabs) Taq DNA polymerase (5 U/ul, New England BioLabs) .. P1-T Adaptor, 15 uM (Life Technologies) 5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′ (41 bp) 5′-TCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGCC-3′ (42 bp) P2-T Adaptor, 15 uM (Life Technologies) 5′-CGCCTTGGCCGTACAGCAGCCTCTTACACAGAGAATGAGGAACCCGGGGCAGTT-3′ (55 bp) 5′-CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT-3′ (53 bp) Library PCR Primer 1, 20 uM (Life Technologies) 5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′ (28 bp) Library PCR Pimer 2, 20 uM (Life Technologies) 5′-CTGCCCCGGGTTCCTCATTCT-3′ (21 bp) Oligonucleotide sequences © Copyright 2008.

    Mutagenesis:

    Article Title:
    Article Snippet: The expression constructs for wild-type SIRT1-EGFP and dominant-negative mutant (H355Y) SIRT1-EGFP were described previously ( ). .. The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio).

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: Primers (forward, 5′-CAAGCTTGCCACCATGTGCTTCCCGAAGGTCCTCTCTGATGACATGAAGAAGCTGAAGG-3′, and reverse, 5′-GTCTAGATCATTTTGAGGATTGGGGTTCAGAGCACTTCTGGGGTGTCAGCTCCTCCTTCTC-3′) were manufactured by Biolegio (Nijmegen, The Netherlands), which contained the RGD to RGE single nucleotide mutation. .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Article Title: Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguini
    Article Snippet: For allelic exchange mutagenesis, linear constructs were created by overlap PCR to replace SK36 genes with the aphA-3 gene (with or without its native promoter) encoding resistance to Kan, as described previously ( ) ( ). .. The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106.

    Article Title: 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation
    Article Snippet: To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S). .. To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S).

    Article Title: Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites
    Article Snippet: The 1.5-kb Kpn I- Bgl II fragment of pLF34 was cloned into pBS-KS (Stratagene); the 3.0-kb Fsp I fragment of this construct was ligated to the 4.7-kb Fsp I fragment of pRS326 ( ) and the 2.6-kb Fsp I fragment of pRS306 ( ) to yield yAR8KG and yAR8KG0, respectively. yAR8KG was isolated from E. coli strain GM2929; the 650-bp Eco RV- Cla I fragment was blunted with T4 polymerase (New England Biolabs [NEB]) plus deoxynucleoside triphosphates (dNTPs) (Roche Molecular Biochemicals), Hin dIII linkers (NEB) were attached, and the fragment was cloned into pRS326 to yield yAR8VCA, which contains the Eco RV- Cla I fragment in the same orientation as in yAR8KG. .. The Eco RV- Cla I fragment contains nucleotides 159457 to 160108 of the chromosome I sequence.

    Aqueous Normal-phase Chromatography:

    Article Title:
    Article Snippet: The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The GATA4-FLAG expression vector was kindly provided by Dr. Mona Nemer (University of Ottawa, Ottawa, Canada).

    Subcloning:

    Article Title:
    Article Snippet: The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The GATA4-FLAG expression vector was kindly provided by Dr. Mona Nemer (University of Ottawa, Ottawa, Canada).

    Purification:

    Article Title: Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
    Article Snippet: The resulting plasmid, pWH1866, was cut with PvuII, and InsTetG− 1 was purified from an agarose gel using the Nucleo Spin Extract kit (Macherey & Nagel, Düren, Germany). .. InsTetG− 1 was obtained from pWH1865 by XbaI restriction, blunt ending using T4-DNA polymerase (NEB, Frankfurt/Main, Germany), and then restriction with HindIII. pCB302b ( ) was digested with AgeI and HindIII and ligated with the InsTetG− 1 fragment to obtain pWH1867.

    Article Title: A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
    Article Snippet: Linearized vector is next purified from agarose gel using the QIAEXII gel extraction kit (Qiagen), and duplicates are pooled. .. For T4 treatment (New England Biolabs), 200 to 400 ng of linearized vector, 4 μL 10× T4 buffer, 4 μL 100 m m dCTP, 2 μL 100 m m dithiothreitol, 0.4 μL bovine serum albumin, 0.8 μL T4 DNA polymerase (New England Biolabs), and water to 40 μL total volume are mixed.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: In the current study, the primers for the second LRP amplification were replaced with WF5T4P (5′-ctc acc tta att aac ata gtg gtc tgc gga acc ggt -3′) and WR55T4P (5′-ata cct ccc ttc tac aat ggc agc cct gcc tcc tct gg -3′). .. An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product. .. Similarly, pCloneK was amplified fully with primers OriFT4P (5′-tat gtt aat taa ggt gag cgg tgt gaa ata ccg cac ag -3) and OriRT4P (5′-att gta gaa ggg agg tat ccg att tcg gcc tat tgg tt -3′).

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Nucleotide sequencing of both strands of purified PCR products was done at the DNA Sequencing Service of Sistemas Genómicos (Valencia, Spain). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Polymerase Chain Reaction:

    Article Title: Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
    Article Snippet: The PCR fragment and pUC18 were digested with PvuII, and the vector was additionally dephosphorylated and ligated. .. InsTetG− 1 was obtained from pWH1865 by XbaI restriction, blunt ending using T4-DNA polymerase (NEB, Frankfurt/Main, Germany), and then restriction with HindIII. pCB302b ( ) was digested with AgeI and HindIII and ligated with the InsTetG− 1 fragment to obtain pWH1867.

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: The PCR protocol comprised incubation for 5 min at 95℃, followed by 27 cycles each consisting of 30 s at 95℃, 30 s at 60℃ or 65℃ and 10 s at 72℃. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Article Title:
    Article Snippet: The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The GATA4-FLAG expression vector was kindly provided by Dr. Mona Nemer (University of Ottawa, Ottawa, Canada).

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: Subsequently, the primers were used to construct the mutants by conventional PCR. .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Article Title: Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguini
    Article Snippet: For allelic exchange mutagenesis, linear constructs were created by overlap PCR to replace SK36 genes with the aphA-3 gene (with or without its native promoter) encoding resistance to Kan, as described previously ( ) ( ). .. The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product. .. Similarly, pCloneK was amplified fully with primers OriFT4P (5′-tat gtt aat taa ggt gag cgg tgt gaa ata ccg cac ag -3) and OriRT4P (5′-att gta gaa ggg agg tat ccg att tcg gcc tat tgg tt -3′).

    Article Title: Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites
    Article Snippet: The 1.5-kb Kpn I- Bgl II fragment of pLF34 was cloned into pBS-KS (Stratagene); the 3.0-kb Fsp I fragment of this construct was ligated to the 4.7-kb Fsp I fragment of pRS326 ( ) and the 2.6-kb Fsp I fragment of pRS306 ( ) to yield yAR8KG and yAR8KG0, respectively. yAR8KG was isolated from E. coli strain GM2929; the 650-bp Eco RV- Cla I fragment was blunted with T4 polymerase (New England Biolabs [NEB]) plus deoxynucleoside triphosphates (dNTPs) (Roche Molecular Biochemicals), Hin dIII linkers (NEB) were attached, and the fragment was cloned into pRS326 to yield yAR8VCA, which contains the Eco RV- Cla I fragment in the same orientation as in yAR8KG. .. The Eco RV- Cla I fragment contains nucleotides 159457 to 160108 of the chromosome I sequence.

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Nucleotide sequencing of both strands of purified PCR products was done at the DNA Sequencing Service of Sistemas Genómicos (Valencia, Spain). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Positron Emission Tomography:

    Article Title: Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene
    Article Snippet: Bacterial strain BL21 (DE3) and plasmid pET-22 b (+) were provided by Institute of Biotechnology, Academy of Military Medical Sciences. .. Restriction enzyme Not I, Nco I and T4 DNA ligase, Vent DNA polymerase, isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from New England Biolabs Company.

    Immunoprecipitation:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: Once again, the same antibodies as were used in the first immunoprecipitation were added into the diluted eluants and incubation, bead binding, washing and elution steps were sequentially repeated. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Functional Asymmetry for the Active Sites of Linked 5-Aminolevulinate Synthase and 8-Amino-7-Oxononanoate Synthase
    Article Snippet: All restriction enzymes, Vent DNA polymerase, and T4 DNA ligase were from New England Biolabs. .. Superdex 200 gel filtration resin was from Amersham Biosciences-GE Healthcare and DNA oligonucleotides were from Integrated DNA Technologies.

    Lysis:

    Article Title: A mutation in the catalytic subunit of yeast telomerase alters primer-template alignment while promoting processivity and protein-DNA binding
    Article Snippet: Genomic DNA was extracted by glass bead lysis. .. RNase-treated DNA samples (~20–40 μg) were blunted by treatment with 4.5 U of T4 DNA polymerase (NEB) in the presence of 1 mM dNTPs at 12°C.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: To detect the site occupied by the RNF2 protein on the human chromatin, the immunoprecipitated DNA fragments were amplified using following primer pairs: F21-17, 5′-ACA CAG TGA GAC TCT GTC TC-3’ and 5′-CGT GTT ACC ATG TCT GTC TAA A-3′; G16-18, 5′-AAG AGG TGC AAC CCA AC-3′ and 5′-GGC AAA ACC CTG TCT CTA CT-3′; F2-23, 5′-TTC TTT CAC TTA AAA GAA AGA CAC-3′ and 5’-TCT AAT ATT AGG GGT GGG AAA-3′; G11-18, 5′-TGG GCC AAG TCC AC-3′ and 5′-TCT CAT CCA AGT CAG GAC A-3′; β-actin, 5′-GCA GGC CTG GCC ATG CG-3′ and 5′-AGT TTT GGC GTT GGC CGC CTT-3′. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Mouse Assay:

    Article Title: Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene
    Article Snippet: Restriction enzyme Not I, Nco I and T4 DNA ligase, Vent DNA polymerase, isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from New England Biolabs Company. .. DNA molecular weight standard λ DNA/ EcoR I + Hind III, goat anti-mouse and goat anti-human IgG-HRP were purchased from Huamei Bioengineering Company and His-Tag precolumn from Invitrogen Company.

    Chromatin Immunoprecipitation:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: Paragraph title: ChIP and ChIP cloning ... The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Article Title: ChIP-exo: A Method to Identify Genomic Location of DNA-binding proteins at Near Single Nucleotide Accuracy
    Article Snippet: Yeast culture .. 37% formaldehyde 2.5 M glycine ST buffer (see recipe) Protease inhibitor cocktail tablets (Roche) FA-lysis buffer (see recipe) 20% SDS (volume/volume) Antibody against the protein of interest Protein A- or G- Sepharose beads or equivalent (e.g. magnetic beads) FA-high salt wash buffer (see recipe) FA-wash 2 buffer (see recipe) FA-wash 3 buffer (see recipe) TE buffer (see recipe) 10 mM Tris-HCl (pH 8.0) NEBuffer 2 (New England BioLabs) 10× BSA (1mg/ml) 3 mM dNTPs T4 DNA polymerase (3 U/ul, New England BioLabs) 10 mM Tris-HCl (pH 7.5) 10× T4 DNA ligase buffer (New England BioLabs) T4 Polynucleotide Kinase (10 U/ul, New England BioLabs) 3 mM dATP Klenow Fragment (3′→5′ exo-) (5 U/ul, New England BioLabs) T4 DNA ligase (500 U/ul, New England BioLabs) 10× phi29 DNA polymerase buffer (New England BioLabs) phi29 DNA polymerase (10 U/ul, New England BioLabs) 10 mM Tris-HCl (pH 9.2) 10× lambda exonuclease buffer (New England BioLabs) Lambda exonulease (5 U/ul, New England BioLabs) RecJf exonuclease (30 U/ul, New England BioLabs) ChIP elution buffer (see recipe) Protease K (20 ug/ul, Roche) 25:24:1 phenol/chloroform/isoamyl alcohol (Sigma) 100% ethanol (Any supplier) 75% (volume/volume) ethanol Glycogen (20 mg/ml, Roche) AMPure magnetic beads (Agencourt) 25 mM dNTPs 10× standard taq reaction buffer (New England BioLabs) Taq DNA polymerase (5 U/ul, New England BioLabs) .. P1-T Adaptor, 15 uM (Life Technologies) 5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3′ (41 bp) 5′-TCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGCC-3′ (42 bp) P2-T Adaptor, 15 uM (Life Technologies) 5′-CGCCTTGGCCGTACAGCAGCCTCTTACACAGAGAATGAGGAACCCGGGGCAGTT-3′ (55 bp) 5′-CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT-3′ (53 bp) Library PCR Primer 1, 20 uM (Life Technologies) 5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′ (28 bp) Library PCR Pimer 2, 20 uM (Life Technologies) 5′-CTGCCCCGGGTTCCTCATTCT-3′ (21 bp) Oligonucleotide sequences © Copyright 2008.

    Plasmid Preparation:

    Article Title: Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
    Article Snippet: The reporter plasmid pWH1867 contains InsTetG− 1 transcriptionally fused to a promoterless lacZ . .. InsTetG− 1 was obtained from pWH1865 by XbaI restriction, blunt ending using T4-DNA polymerase (NEB, Frankfurt/Main, Germany), and then restriction with HindIII. pCB302b ( ) was digested with AgeI and HindIII and ligated with the InsTetG− 1 fragment to obtain pWH1867.

    Article Title: A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
    Article Snippet: The pellet is next dried and resuspended in 50 μL of water (at 50°C for 5 to 10 min). .. For T4 treatment (New England Biolabs), 200 to 400 ng of linearized vector, 4 μL 10× T4 buffer, 4 μL 100 m m dCTP, 2 μL 100 m m dithiothreitol, 0.4 μL bovine serum albumin, 0.8 μL T4 DNA polymerase (New England Biolabs), and water to 40 μL total volume are mixed. .. The mixture is centrifuged at maximum speed for 1 min, incubated at 22°C for at least 30 min (up to 2 h), inactivated at 75°C for 20 min, and centrifuged again at maximum speed for 1 min. T4 treated vectors can be stored at 4°C until further use.

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: The cross-links were reversed, and the samples were sequentially treated with RNase A and proteinase K, and then isolated as performed in the ChIP assay. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector. .. To determine the location and flanking genes on the RNF2-responding loci on the chromosomes, a Human BLAST Search of UCSC Genome Bioinformatics ( ) and the BLAST of NCBI ( ) was used.

    Article Title:
    Article Snippet: To generate the blank control vector of p300-HA (CMVβ(Δp300)), the p300-HA plasmid was digested with NotI and HindIII. .. The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio). .. The FLAG-PCAF (p300/CBP-associated factor) expression vector was kindly provided by Dr. Yoshihiro Nakatani (Dana Farber Cancer Institute, Boston).

    Article Title: Use of single stranded targeting DNA or negative selection does not further increase the efficiency of a GGTA1 promoter trap
    Article Snippet: After modifications to cDNA, clone 3460621 were performed to remove vector backbone restriction sites, the resulting plasmid was named pBB2. .. To isolate the CAG promoter, pCAG-Cre:GFP (Addgene plasmid 13776; [ ]) was cut with EcoRI and SalI, and the 3` ends were extended with T4 DNA polymerase (New England Biolabs), to produce blunt ends. .. This fragment was subcloned into pBB2, between the AattII and EcoRI restriction sites that had been digested, and then treated with T4 DNA polymerase (New England Biolabs), to produce blunt ends.

    Article Title: Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguini
    Article Snippet: We began with plasmid pJFP96, which contains 1.35 kb of S. sanguinis DNA encompassing the SSA_0169 gene and flanking sequences, with the SSA_0169 gene interrupted by the aad9 gene encoding resistance to Spc.4 We first deleted HindIII and SphI restriction sites to facilitate downstream cloning reactions. .. The deletions were accomplished by digesting pJFP96 with HindIII and SphI (New England Biolabs), followed by treatment with T4 DNA polymerase (New England Biolabs) to create blunt ends, and ligating the blunt ends to re-circularize the plasmid using T4 DNA ligase (Invitrogen), resulting in creation of pJFP106. .. Next, an EcoRI to BamHI fragment of pDR111 (kindly provided by Dr. David Rudner, Harvard Medical School) containing the Phyper-spank lacZo lacI expression cassette was amplified by PCR using primers that replaced the EcoRI and BamHI restriction sites with NotI and AscI sites, respectively.

    Article Title: Comprehensive Cloning of Patient-derived 9022-bp Amplicons of Hepatitis C Virus
    Article Snippet: An aliquot of 2.28 μg (~0.5 pmol) purified LRP product mixed with 5 U of T4 DNA polymerase (New England Biolabs) in the presence of 2.5 mM dCTP (Invitrogen), incubated at room temperature for 30 minutes followed by heat inactivation for 20 minutes, which generated 18-bp sticky ends on the LRP product. .. The resulting PCR product was treated with T4 DNA polymerase in the presence of dGTP, which created 18-bp sticky ends matching to the one from the LRP amplicon.

    Article Title: 3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation
    Article Snippet: The synthetic sequences were synthesized and subcloned into pIDTSMART plasmids by Integrated DNA Technologies (IDT). .. To replace the Alu Jb repeat in plasmid p Alu IR with a synthetic DNA fragment, p Alu IR was first linearized with SpeI and blunt-ended with T4 DNA polymerase (New England Biolabs, cat. no. M0203S). .. Linearized p Alu IR was then digested with SbfI to remove the Alu Jb repeat.

    Article Title: Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites
    Article Snippet: The 1.5-kb Kpn I- Bgl II fragment of pLF34 was cloned into pBS-KS (Stratagene); the 3.0-kb Fsp I fragment of this construct was ligated to the 4.7-kb Fsp I fragment of pRS326 ( ) and the 2.6-kb Fsp I fragment of pRS306 ( ) to yield yAR8KG and yAR8KG0, respectively. yAR8KG was isolated from E. coli strain GM2929; the 650-bp Eco RV- Cla I fragment was blunted with T4 polymerase (New England Biolabs [NEB]) plus deoxynucleoside triphosphates (dNTPs) (Roche Molecular Biochemicals), Hin dIII linkers (NEB) were attached, and the fragment was cloned into pRS326 to yield yAR8VCA, which contains the Eco RV- Cla I fragment in the same orientation as in yAR8KG. .. The mutation in the 11 of 11 match to the ACS was made as described by Kunkel , and the mutation in the 9 of 11 match was made by fusion PCR ( ).

    Article Title: Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans
    Article Snippet: Restriction endonucleases, T4 DNA ligase, alkaline phosphatase, and the Klenow fragment of DNA polymerase I were purchased from New England Biolabs (Beverly, Mass.) and used as recommended by the manufacturer. .. PCR products were purified by using the QIAquick PCR purification system (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

    Dominant Negative Mutation:

    Article Title:
    Article Snippet: The expression constructs for wild-type SIRT1-EGFP and dominant-negative mutant (H355Y) SIRT1-EGFP were described previously ( ). .. The vector overhangs were then blunted by using T4 DNA polymerase (New England Biolabs), and the blunt ends were ligated with the DNA ligation kit Mighty Mix (Takara-Bio).

    Recombinant:

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Total genomic DNA from recombinant yeasts was isolated using the Wizard DNA Purification kit (Promega Corporation, Madison, WI). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    Agarose Gel Electrophoresis:

    Article Title: Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
    Article Snippet: The resulting plasmid, pWH1866, was cut with PvuII, and InsTetG− 1 was purified from an agarose gel using the Nucleo Spin Extract kit (Macherey & Nagel, Düren, Germany). .. InsTetG− 1 was obtained from pWH1865 by XbaI restriction, blunt ending using T4-DNA polymerase (NEB, Frankfurt/Main, Germany), and then restriction with HindIII. pCB302b ( ) was digested with AgeI and HindIII and ligated with the InsTetG− 1 fragment to obtain pWH1867.

    Article Title: A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
    Article Snippet: Linearized vector is next purified from agarose gel using the QIAEXII gel extraction kit (Qiagen), and duplicates are pooled. .. For T4 treatment (New England Biolabs), 200 to 400 ng of linearized vector, 4 μL 10× T4 buffer, 4 μL 100 m m dCTP, 2 μL 100 m m dithiothreitol, 0.4 μL bovine serum albumin, 0.8 μL T4 DNA polymerase (New England Biolabs), and water to 40 μL total volume are mixed.

    Produced:

    Article Title: Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1
    Article Snippet: The RGD deletion mutants were produced by digesting the pCDNA3-IL-32β/IL-32γ expression plasmids with HindIII (New England Biolabs, Ipswich, MA) and XbaI (New England Biolabs) to separate the backbone and insert. .. Next, the BtgI and XbaI sites were both treated with T4 DNA polymerase (New England Biolabs) together with 100 μ m dNTPs to create blunt ends to facilitate ligation of both ends with T4 DNA ligase (New England Biolabs).

    Article Title: Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene
    Article Snippet: Restriction enzyme Not I, Nco I and T4 DNA ligase, Vent DNA polymerase, isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from New England Biolabs Company. .. Restriction enzyme Not I, Nco I and T4 DNA ligase, Vent DNA polymerase, isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from New England Biolabs Company.

    Concentration Assay:

    Article Title: Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
    Article Snippet: The DNA concentration was determined from the absorption at 260 nm. .. InsTetG− 1 was obtained from pWH1865 by XbaI restriction, blunt ending using T4-DNA polymerase (NEB, Frankfurt/Main, Germany), and then restriction with HindIII. pCB302b ( ) was digested with AgeI and HindIII and ligated with the InsTetG− 1 fragment to obtain pWH1867.

    DNA Purification:

    Article Title: Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50
    Article Snippet: Total genomic DNA from recombinant yeasts was isolated using the Wizard DNA Purification kit (Promega Corporation, Madison, WI). .. Platinum Taq polymerase (Invitrogen), DNA restriction enzymes (New England Biolabs Inc., Beverly, MA), and T4 DNA ligase (Promega) were used as suggested by the manufacturers.

    CTG Assay:

    Article Title: Identification and Characterization of RNF2 Response Elements in Human Kidney Cells
    Article Snippet: To detect the site occupied by the RNF2 protein on the human chromatin, the immunoprecipitated DNA fragments were amplified using following primer pairs: F21-17, 5′-ACA CAG TGA GAC TCT GTC TC-3’ and 5′-CGT GTT ACC ATG TCT GTC TAA A-3′; G16-18, 5′-AAG AGG TGC AAC CCA AC-3′ and 5′-GGC AAA ACC CTG TCT CTA CT-3′; F2-23, 5′-TTC TTT CAC TTA AAA GAA AGA CAC-3′ and 5’-TCT AAT ATT AGG GGT GGG AAA-3′; G11-18, 5′-TGG GCC AAG TCC AC-3′ and 5′-TCT CAT CCA AGT CAG GAC A-3′; β-actin, 5′-GCA GGC CTG GCC ATG CG-3′ and 5′-AGT TTT GGC GTT GGC CGC CTT-3′. .. The isolated DNA fragments were blunted with T4 DNA polymerase (NEB), and cloned into the HindV site of the pBluescript II vector.

    Gel Extraction:

    Article Title: A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants
    Article Snippet: Linearized vector is next purified from agarose gel using the QIAEXII gel extraction kit (Qiagen), and duplicates are pooled. .. For T4 treatment (New England Biolabs), 200 to 400 ng of linearized vector, 4 μL 10× T4 buffer, 4 μL 100 m m dCTP, 2 μL 100 m m dithiothreitol, 0.4 μL bovine serum albumin, 0.8 μL T4 DNA polymerase (New England Biolabs), and water to 40 μL total volume are mixed.

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  • 99
    New England Biolabs t4 dna polymerase
    Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2019-12
    99/100 stars
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    99
    New England Biolabs t4 dna ligase
    Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat  = 2.2 ± 0.2 × 10 −4  s −1  and K M  = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0  was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat  is 8 × 10 −3  s −1  with an upper threshold for the K M  estimated to be 1 nM.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2019-12
    99/100 stars
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    Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Clone Assay, Activity Assay, Polymerase Chain Reaction, Sequencing, Incubation, Plasmid Preparation

    Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme  Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent  E. coli  DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Amplification, Clone Assay, Purification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay

    Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C ,  D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C , D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Incubation, Agarose Gel Electrophoresis

    Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Activity Assay, Plasmid Preparation, Incubation, Generated, Agarose Gel Electrophoresis

    Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat  = 2.2 ± 0.2 × 10 −4  s −1  and K M  = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0  was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat  is 8 × 10 −3  s −1  with an upper threshold for the K M  estimated to be 1 nM.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat = 2.2 ± 0.2 × 10 −4 s −1 and K M = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0 was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat is 8 × 10 −3 s −1 with an upper threshold for the K M estimated to be 1 nM.

    Article Snippet: High concentration T4 DNA ligase was obtained from NEB; concentration and adenylylation state were determined as previously described ( ).

    Techniques: Ligation, Concentration Assay

    Ligation of DNA splinted by RNA. (Left) Outline of the ligation assay: a 5′-phosphorylated, 3′-FAM labelled DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor’ oligonucleotide are annealed to a complementary RNA or DNA splint. This substrate was reacted with a ligase to form a mixture of unreacted starting material, adenylylated DNA and ligated product. The products were denatured, separated on CE, and detected by fluorescence. (Right) ligation of the standard RNA-splinted substrate in ligase assay buffer for 15 min at 25°C with ( A ) no enzyme, ( B ) 1 µM T4 DNA ligase and 10 µM ATP, ( C ) 1 µM T4 DNA ligase and 1 mM ATP, ( D ) 100 nM PBCV-1 DNA ligase and 10 µM ATP, and ( E ) 100 nM PBCV-1 DNA ligase and 1 mM ATP. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by coelution with synthetically prepared standards.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Ligation of DNA splinted by RNA. (Left) Outline of the ligation assay: a 5′-phosphorylated, 3′-FAM labelled DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor’ oligonucleotide are annealed to a complementary RNA or DNA splint. This substrate was reacted with a ligase to form a mixture of unreacted starting material, adenylylated DNA and ligated product. The products were denatured, separated on CE, and detected by fluorescence. (Right) ligation of the standard RNA-splinted substrate in ligase assay buffer for 15 min at 25°C with ( A ) no enzyme, ( B ) 1 µM T4 DNA ligase and 10 µM ATP, ( C ) 1 µM T4 DNA ligase and 1 mM ATP, ( D ) 100 nM PBCV-1 DNA ligase and 10 µM ATP, and ( E ) 100 nM PBCV-1 DNA ligase and 1 mM ATP. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by coelution with synthetically prepared standards.

    Article Snippet: High concentration T4 DNA ligase was obtained from NEB; concentration and adenylylation state were determined as previously described ( ).

    Techniques: Ligation, Fluorescence

    Detection of defined amounts of luciferase mRNA from a mixture with Jurkat total RNA through ligation of specific DNA probes (Probe set A) and detection by qPCR. RNA/DNA probe mixtures were annealed then incubated with either T4 DNA ligase (open circle) or PBCV-1 DNA ligase (×). The qPCR C q  for each experiment was recorded, with lower C q  indicating higher concentration of ligated probes. ( A ) Ligation time course with either 1 (solid line) or 0.01 ng (dotted line) of luciferase mRNA target, 0–8 h ligation time. ( B ) Dependence of C q  after probe ligation with target luciferase mRNA present over a 7 log concentration range using a 2 h ligation time. NTC = no template control. The error bars show one standard deviation of the average of a minimum of three replicates.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Detection of defined amounts of luciferase mRNA from a mixture with Jurkat total RNA through ligation of specific DNA probes (Probe set A) and detection by qPCR. RNA/DNA probe mixtures were annealed then incubated with either T4 DNA ligase (open circle) or PBCV-1 DNA ligase (×). The qPCR C q for each experiment was recorded, with lower C q indicating higher concentration of ligated probes. ( A ) Ligation time course with either 1 (solid line) or 0.01 ng (dotted line) of luciferase mRNA target, 0–8 h ligation time. ( B ) Dependence of C q after probe ligation with target luciferase mRNA present over a 7 log concentration range using a 2 h ligation time. NTC = no template control. The error bars show one standard deviation of the average of a minimum of three replicates.

    Article Snippet: High concentration T4 DNA ligase was obtained from NEB; concentration and adenylylation state were determined as previously described ( ).

    Techniques: Luciferase, Ligation, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay, Standard Deviation

    Ligation of RNA-splinted DNA substrates by PBCV-1 and T4 DNA ligases under general reaction conditions. The 16 RNA-splinted DNA substrates, representing all possible base pairs at the ligation junction, were reacted and the extent of ligation and abortive adenylylation measured. The bases listed on the X axis (dN/pdN) refer to the identity of the base of the DNA acceptor at the ligation junction (dN) and the identity of the phosphorylated base on the donor at the ligation junction (pdN). For all substrates, the correct Watson–Crick base-pairing partner was present in the RNA splint. All reactions were incubated at 37°C in 50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 nM RNA-splinted DNA substrate and ( A ) 100 nM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( B ) 100 nM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( C ) 1 µM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( D ) 1 µM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( E ) 1 µM T4 DNA ligase and 10 µM ATP for 15 min; and ( F ) 1 µM T4 DNA ligase and 10 µM ATP for 4 h. Here the total height of the bar indicates the fraction of starting material converted to products, with the solid portion indicating ligation product yield and the hashed portion indicating AppDNA yield.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Ligation of RNA-splinted DNA substrates by PBCV-1 and T4 DNA ligases under general reaction conditions. The 16 RNA-splinted DNA substrates, representing all possible base pairs at the ligation junction, were reacted and the extent of ligation and abortive adenylylation measured. The bases listed on the X axis (dN/pdN) refer to the identity of the base of the DNA acceptor at the ligation junction (dN) and the identity of the phosphorylated base on the donor at the ligation junction (pdN). For all substrates, the correct Watson–Crick base-pairing partner was present in the RNA splint. All reactions were incubated at 37°C in 50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 nM RNA-splinted DNA substrate and ( A ) 100 nM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( B ) 100 nM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( C ) 1 µM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( D ) 1 µM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( E ) 1 µM T4 DNA ligase and 10 µM ATP for 15 min; and ( F ) 1 µM T4 DNA ligase and 10 µM ATP for 4 h. Here the total height of the bar indicates the fraction of starting material converted to products, with the solid portion indicating ligation product yield and the hashed portion indicating AppDNA yield.

    Article Snippet: High concentration T4 DNA ligase was obtained from NEB; concentration and adenylylation state were determined as previously described ( ).

    Techniques: Ligation, Dominant Negative Mutation, Incubation