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    New England Biolabs t4 polynucleotide kinase
    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using <t>T4</t> polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs m0203l t4 dna polymerase kinase new england biolabs
    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized <t>T4</t> DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    M0203l T4 Dna Polymerase Kinase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs m0202l t4 dna polymerase new england biolabs
    Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and <t>T4-Ligase.</t> Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.
    M0202l T4 Dna Polymerase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs b0202 q5 high fidelity dna polymerase new england biolabs
    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, <t>T4</t> ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    B0202 Q5 High Fidelity Dna Polymerase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Journal: Cancers

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    doi: 10.3390/cancers6031464

    Figure Lengend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Article Snippet: The purified probe (100 ng) was then 5' labeled with [32 P-γ] ATP with T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Labeling, Incubation, Negative Control

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation

    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

    Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

    Journal: PLoS ONE

    Article Title: A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

    doi: 10.1371/journal.pone.0109196

    Figure Lengend Snippet: Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

    Article Snippet: Meanwhile a second reaction for the ligation was prepared. (II) 3 µL 10 mM ATP, 1 µL 10 x Tango Buffer, 1 µL 10 mM DTT, 1 µL T4-Ligase (NEB) addition of H2 0 to reach 10 µL.

    Techniques: Clone Assay, Amplification, Introduce, Incubation, Transformation Assay, Plasmid Preparation, Construct, Marker, Selection, Sequencing

    Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 5′ overhang protocol through the ligation to dsDNA fragments. In all gels, the left lane shows a DNA ladder with molecule lengths indicated in kb. ( A ) Result of the transcript reaction of a 4.2 kb dsRNA molecule with 5′ overhangs complementary to those of 0.4 kb dsDNA molecules: the more slowly migrating band only appears following transcription. ( B ) The transcription reaction following treatment with DNase I. The single remaining band must be dsRNA. ( C ) Ligation by T4 DNA ligase of the 4.2 kb dsRNA molecule with 5′ overhangs to 0.4 kb dsDNA ends tagged with biotin and digoxigenin. The dsDNA molecules were digested with BsmA1 to yield overhangs complementary to those incorporated into the 4.2 kb dsRNA molecule. The appearance of a new more slowly migrating band indicates the presence of molecules that have been ligated using the designed 5′ overhangs.

    Journal: Nucleic Acids Research

    Article Title: Joining of long double-stranded RNA molecules through controlled overhangs

    doi: 10.1093/nar/gnh138

    Figure Lengend Snippet: Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 5′ overhang protocol through the ligation to dsDNA fragments. In all gels, the left lane shows a DNA ladder with molecule lengths indicated in kb. ( A ) Result of the transcript reaction of a 4.2 kb dsRNA molecule with 5′ overhangs complementary to those of 0.4 kb dsDNA molecules: the more slowly migrating band only appears following transcription. ( B ) The transcription reaction following treatment with DNase I. The single remaining band must be dsRNA. ( C ) Ligation by T4 DNA ligase of the 4.2 kb dsRNA molecule with 5′ overhangs to 0.4 kb dsDNA ends tagged with biotin and digoxigenin. The dsDNA molecules were digested with BsmA1 to yield overhangs complementary to those incorporated into the 4.2 kb dsRNA molecule. The appearance of a new more slowly migrating band indicates the presence of molecules that have been ligated using the designed 5′ overhangs.

    Article Snippet: The dsRNA–dsRNA ligation reactions (total volume 20 μl) using T4 DNA ligase consisted of up to 100–500 ng 4.2 dsRNA, a 5-fold molar excess of both 0.4 kb dsRNAs, Quick Ligase buffer and 2 μl T4 DNA Quick Ligase (New England Biolabs), and 40 U of Protector RNase Inhibitor (Roche Applied Science, Germany).

    Techniques: Nucleic Acid Electrophoresis, Ligation

    Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 5′ overhang protocol through the ligation to dsRNA fragments. In all gels, the left lane shows a DNA ladder with molecule lengths indicated in kb. ( A ) Left lane: results of a transcription reaction on two separate 4.2 kb PCR products: the more slowly migrating band only appears following transcription. Right lane: the same reaction following treatment with DNase I. The band corresponding to the PCR products has been eliminated by DNase digestion, leaving a 4.2 kb dsRNA molecule. ( B ) The 4.2 kb dsRNA molecule ligated by T4 DNA ligase to 0.4 kb dsRNA fragments, all with 5′ overhangs. Two additional, more slowly migrating bands have appeared, corresponding to ligation products. ( C ) Controls applied to distinguish hybridization from ligation using T4 DNA ligase. We carried out two separate reactions, one reaction in the absence of ligase, and another in the presence of ligase. Both cases result in a gel pattern similar to (B) prior to heating. However, following heating of the reaction in the absence of ligase (left lane), only the band corresponding to the 4.2 kb dsRNA molecule remained, whereas following heating of the reaction in the presence of ligase (right lane), both the 4.2 kb dsRNA band and a longer band remained. This demonstrated that ligase repaired the phosphodiester backbone. We estimate that approximately 10% of the 4.2 kb dsRNA molecules were ligated to at least one of the shorter fragments. ( D ) The same experiment as in (C), but using Rnl2. The reaction was incubated for 30 min, heated to 55°C (well above the T m ≈ 28°C for the 8 nt overhangs) for 15 min, rapidly chilled on ice, and run on gel. Left lane: in the absence of Rnl2; right lane: in the presence of Rnl2. Again, the fact that longer bands remain in the right lane following heating indicates the presence of ligation products. The use of Rnl2 appears to increase the ligation efficiency to ≥50%.

    Journal: Nucleic Acids Research

    Article Title: Joining of long double-stranded RNA molecules through controlled overhangs

    doi: 10.1093/nar/gnh138

    Figure Lengend Snippet: Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 5′ overhang protocol through the ligation to dsRNA fragments. In all gels, the left lane shows a DNA ladder with molecule lengths indicated in kb. ( A ) Left lane: results of a transcription reaction on two separate 4.2 kb PCR products: the more slowly migrating band only appears following transcription. Right lane: the same reaction following treatment with DNase I. The band corresponding to the PCR products has been eliminated by DNase digestion, leaving a 4.2 kb dsRNA molecule. ( B ) The 4.2 kb dsRNA molecule ligated by T4 DNA ligase to 0.4 kb dsRNA fragments, all with 5′ overhangs. Two additional, more slowly migrating bands have appeared, corresponding to ligation products. ( C ) Controls applied to distinguish hybridization from ligation using T4 DNA ligase. We carried out two separate reactions, one reaction in the absence of ligase, and another in the presence of ligase. Both cases result in a gel pattern similar to (B) prior to heating. However, following heating of the reaction in the absence of ligase (left lane), only the band corresponding to the 4.2 kb dsRNA molecule remained, whereas following heating of the reaction in the presence of ligase (right lane), both the 4.2 kb dsRNA band and a longer band remained. This demonstrated that ligase repaired the phosphodiester backbone. We estimate that approximately 10% of the 4.2 kb dsRNA molecules were ligated to at least one of the shorter fragments. ( D ) The same experiment as in (C), but using Rnl2. The reaction was incubated for 30 min, heated to 55°C (well above the T m ≈ 28°C for the 8 nt overhangs) for 15 min, rapidly chilled on ice, and run on gel. Left lane: in the absence of Rnl2; right lane: in the presence of Rnl2. Again, the fact that longer bands remain in the right lane following heating indicates the presence of ligation products. The use of Rnl2 appears to increase the ligation efficiency to ≥50%.

    Article Snippet: The dsRNA–dsRNA ligation reactions (total volume 20 μl) using T4 DNA ligase consisted of up to 100–500 ng 4.2 dsRNA, a 5-fold molar excess of both 0.4 kb dsRNAs, Quick Ligase buffer and 2 μl T4 DNA Quick Ligase (New England Biolabs), and 40 U of Protector RNase Inhibitor (Roche Applied Science, Germany).

    Techniques: Nucleic Acid Electrophoresis, Ligation, Polymerase Chain Reaction, Hybridization, Incubation

    Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 3′ overhang protocol through the ligation to dsDNA fragments. In all gels, the left lane shows a dsDNA ladder with molecule lengths indicated in kb. ( A ) The direct outcome of the transcription reaction on two 7.4 kb PCR templates: the more slowly migrating band only appears following transcription. ( B ) The transcription reaction after treatment with DNase I: the PCR products have disappeared, leaving a single band which must be dsRNA. ( C ) Test of the incorporation of overhangs through the ligation of the 7.4 kb dsRNA product to two distinct 0.4 kb dsDNA fragments (labelled with biotin and digoxigenin, respectively) by T4 DNA ligase. The dsDNA ends have been digested with KpnI and SacI. A new band appears that migrates more slowly on gel, corresponding to molecules that have been ligated using the designed 3′ overhangs.

    Journal: Nucleic Acids Research

    Article Title: Joining of long double-stranded RNA molecules through controlled overhangs

    doi: 10.1093/nar/gnh138

    Figure Lengend Snippet: Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 3′ overhang protocol through the ligation to dsDNA fragments. In all gels, the left lane shows a dsDNA ladder with molecule lengths indicated in kb. ( A ) The direct outcome of the transcription reaction on two 7.4 kb PCR templates: the more slowly migrating band only appears following transcription. ( B ) The transcription reaction after treatment with DNase I: the PCR products have disappeared, leaving a single band which must be dsRNA. ( C ) Test of the incorporation of overhangs through the ligation of the 7.4 kb dsRNA product to two distinct 0.4 kb dsDNA fragments (labelled with biotin and digoxigenin, respectively) by T4 DNA ligase. The dsDNA ends have been digested with KpnI and SacI. A new band appears that migrates more slowly on gel, corresponding to molecules that have been ligated using the designed 3′ overhangs.

    Article Snippet: The dsRNA–dsRNA ligation reactions (total volume 20 μl) using T4 DNA ligase consisted of up to 100–500 ng 4.2 dsRNA, a 5-fold molar excess of both 0.4 kb dsRNAs, Quick Ligase buffer and 2 μl T4 DNA Quick Ligase (New England Biolabs), and 40 U of Protector RNase Inhibitor (Roche Applied Science, Germany).

    Techniques: Nucleic Acid Electrophoresis, Ligation, Polymerase Chain Reaction

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Journal: Langmuir : the ACS journal of surfaces and colloids

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    doi: 10.1021/acs.langmuir.8b01812

    Figure Lengend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Article Snippet: Annealed circles were ligated with the addition of 1 μ L of T4 DNA ligase (NEB M0202S) at room temperature for ~5 h. Ligated circles can be stored at 4 °C for up to 1 month.

    Techniques: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Journal: Nucleic Acids Research

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    doi:

    Figure Lengend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Article Snippet: The reaction was incubated for 10 min at 37°C in T4 ligase buffer (New England Biolabs) containing 100 µg/ml bovine serum albumin, 75 mM KCl and the heteroduplex oligo recovered after Dpn II digestion (estimated to be a ∼100-fold molar excess over the plasmid ends).

    Techniques: Plasmid Preparation, Ligation, Generated