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  • 99
    New England Biolabs t 4 polynucleotide kinase
    T 4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t 4 dna ligase
    T 4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa t 4 dna ligase
    T 4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t 4 dna ligase
    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.
    T 4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher t 4 kinase
    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.
    T 4 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Frontier Scientific h2 t 4 pyp
    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.
    H2 T 4 Pyp, supplied by Frontier Scientific, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs t4 pdg
    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by <t>T4-PDG.</t> Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively
    T4 Pdg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa t4 ligase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 909 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher t4 dna polymerase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using <t>T4</t> DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 2061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polymerase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using <t>T4</t> DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 657 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore t4 beta glucosyltransferase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using <t>T4</t> DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Beta Glucosyltransferase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 kinase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using <t>T4</t> DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher t4 β glucosyltransferase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using <t>T4</t> DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 β Glucosyltransferase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson rpa t4
    Design and testing of a dual reporter assay measuring inhibition efficiency and specificity. A. A dual reporter system was designed by incorporating the R-Luc gene, under the control of a tTA-responsive promoter, into effector cells. When these cells are fused to target cells, two readouts are measured sequentially: F-Luc activity measures the extent of cell-cell fusion and R-Luc activity measures any off-target effect. Specific fusion inhibitors are expected to give low F-Luc and high R-Luc activities, whereas unrelated inhibition results in low activities of both luciferases. B. Various compounds were tested with the dual reporter system using H-AD8#15Ren effector cells and CEM-R5L1#21 target cells. Both F-Luc and R-Luc activities were measured and normalized to those activities seen in cells without any inhibitor. The final concentration of DMSO during the cell-cell fusion assay was 0.1% unless otherwise indicated. T20, T20 peptide (118 nM); Mar, Maraviroc (1.18 µM); CHX, cycloheximide (100 µg/ml); <t>RPA,</t> <t>RPA-T4</t> (anti-CD4 antibody, 1 nM); OKT, OKT4 (anti-CD4 antibody, 1 nM); Con.Ab, Control antibody (1 nM); NBD, NBD-556 (9.1 µM); Dox, doxycycline (2 µg/ml); EF, efavirenz (an HIV-1 reverse transcriptase inhibitor, 312 nM).
    Rpa T4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad rpa t4
    Design and testing of a dual reporter assay measuring inhibition efficiency and specificity. A. A dual reporter system was designed by incorporating the R-Luc gene, under the control of a tTA-responsive promoter, into effector cells. When these cells are fused to target cells, two readouts are measured sequentially: F-Luc activity measures the extent of cell-cell fusion and R-Luc activity measures any off-target effect. Specific fusion inhibitors are expected to give low F-Luc and high R-Luc activities, whereas unrelated inhibition results in low activities of both luciferases. B. Various compounds were tested with the dual reporter system using H-AD8#15Ren effector cells and CEM-R5L1#21 target cells. Both F-Luc and R-Luc activities were measured and normalized to those activities seen in cells without any inhibitor. The final concentration of DMSO during the cell-cell fusion assay was 0.1% unless otherwise indicated. T20, T20 peptide (118 nM); Mar, Maraviroc (1.18 µM); CHX, cycloheximide (100 µg/ml); <t>RPA,</t> <t>RPA-T4</t> (anti-CD4 antibody, 1 nM); OKT, OKT4 (anti-CD4 antibody, 1 nM); Con.Ab, Control antibody (1 nM); NBD, NBD-556 (9.1 µM); Dox, doxycycline (2 µg/ml); EF, efavirenz (an HIV-1 reverse transcriptase inhibitor, 312 nM).
    Rpa T4, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher rpa t4
    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs <t>RPA-T4,</t> SK3 and #21, or mouse IgG as
    Rpa T4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Nexcelom Bioscience t4 cellometer
    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs <t>RPA-T4,</t> SK3 and #21, or mouse IgG as
    T4 Cellometer, supplied by Nexcelom Bioscience, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher t4 pol
    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs <t>RPA-T4,</t> SK3 and #21, or mouse IgG as
    T4 Pol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Toyobo t4 polymerase
    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs <t>RPA-T4,</t> SK3 and #21, or mouse IgG as
    T4 Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Journal: Nature Communications

    Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions

    doi: 10.1038/s41467-019-09146-5

    Figure Lengend Snippet: In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Article Snippet: We confirmed the existence of these adducts by the sensitivity of this damaged template to an enzyme called, T4-PDG (from bacteriophage T4, pyrimidine dimer glycosylase; NEB) (Fig. , third lane).

    Techniques: In Vitro, Generated, Nucleic Acid Electrophoresis, Irradiation, Labeling

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Article Snippet: Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Journal: Nucleic Acids Research

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

    doi: 10.1093/nar/gkq572

    Figure Lengend Snippet: Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Article Snippet: PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP.

    Techniques: Ligation, Purification, Sequencing, Blocking Assay

    Design and testing of a dual reporter assay measuring inhibition efficiency and specificity. A. A dual reporter system was designed by incorporating the R-Luc gene, under the control of a tTA-responsive promoter, into effector cells. When these cells are fused to target cells, two readouts are measured sequentially: F-Luc activity measures the extent of cell-cell fusion and R-Luc activity measures any off-target effect. Specific fusion inhibitors are expected to give low F-Luc and high R-Luc activities, whereas unrelated inhibition results in low activities of both luciferases. B. Various compounds were tested with the dual reporter system using H-AD8#15Ren effector cells and CEM-R5L1#21 target cells. Both F-Luc and R-Luc activities were measured and normalized to those activities seen in cells without any inhibitor. The final concentration of DMSO during the cell-cell fusion assay was 0.1% unless otherwise indicated. T20, T20 peptide (118 nM); Mar, Maraviroc (1.18 µM); CHX, cycloheximide (100 µg/ml); RPA, RPA-T4 (anti-CD4 antibody, 1 nM); OKT, OKT4 (anti-CD4 antibody, 1 nM); Con.Ab, Control antibody (1 nM); NBD, NBD-556 (9.1 µM); Dox, doxycycline (2 µg/ml); EF, efavirenz (an HIV-1 reverse transcriptase inhibitor, 312 nM).

    Journal: PLoS ONE

    Article Title: An Inducible Cell-Cell Fusion System with Integrated Ability to Measure the Efficiency and Specificity of HIV-1 Entry Inhibitors

    doi: 10.1371/journal.pone.0026731

    Figure Lengend Snippet: Design and testing of a dual reporter assay measuring inhibition efficiency and specificity. A. A dual reporter system was designed by incorporating the R-Luc gene, under the control of a tTA-responsive promoter, into effector cells. When these cells are fused to target cells, two readouts are measured sequentially: F-Luc activity measures the extent of cell-cell fusion and R-Luc activity measures any off-target effect. Specific fusion inhibitors are expected to give low F-Luc and high R-Luc activities, whereas unrelated inhibition results in low activities of both luciferases. B. Various compounds were tested with the dual reporter system using H-AD8#15Ren effector cells and CEM-R5L1#21 target cells. Both F-Luc and R-Luc activities were measured and normalized to those activities seen in cells without any inhibitor. The final concentration of DMSO during the cell-cell fusion assay was 0.1% unless otherwise indicated. T20, T20 peptide (118 nM); Mar, Maraviroc (1.18 µM); CHX, cycloheximide (100 µg/ml); RPA, RPA-T4 (anti-CD4 antibody, 1 nM); OKT, OKT4 (anti-CD4 antibody, 1 nM); Con.Ab, Control antibody (1 nM); NBD, NBD-556 (9.1 µM); Dox, doxycycline (2 µg/ml); EF, efavirenz (an HIV-1 reverse transcriptase inhibitor, 312 nM).

    Article Snippet: RPA-T4, 2D7 and control isotype antibodies were purchased from BD Biosciences; OKT4 was obtained from eBiosciences.

    Techniques: Reporter Assay, Inhibition, Activity Assay, Concentration Assay, Cell-Cell Fusion Assay, Recombinase Polymerase Amplification

    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs RPA-T4, SK3 and #21, or mouse IgG as

    Journal:

    Article Title: Interaction with CD4 and Antibodies to CD4-Induced Epitopes of the Envelope gp120 from a Microglial Cell-Adapted Human Immunodeficiency Virus Type 1 Isolate

    doi: 10.1128/JVI.79.11.6703-6713.2005

    Figure Lengend Snippet: Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs RPA-T4, SK3 and #21, or mouse IgG as

    Article Snippet: HOS cells stably expressing CD4 and CCR5 (plated at 104 cells/well into 96-well plates) were incubated for 1 h at 4°C with medium alone or in medium with anti-CD4 MAbs #21, RPA-T4 (eBioscience, San Diego, CA), SK3 (BD Biosciences), or mouse IgG and infected with pseudotypes for 6 h. Luciferase activity was measured at 2 to 3 days postinfection.

    Techniques: Inhibition, Infection, Luciferase, Recombinase Polymerase Amplification