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    New England Biolabs m0203l t4 dna polymerase kinase new england biolabs
    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 <t>T4</t> 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.
    M0203l T4 Dna Polymerase Kinase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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 5308 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
    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.
    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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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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    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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-34580-8

    Figure Lengend 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.

    Article Snippet: The sequencing adaptor oligos were: 5′-CCTCTCTATGGGCAGTCGGTGATTTTTTTT-3′ (universal adaptor) and 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG NNNNNNNNNN TTTTTTTT-3′ (barcoded adaptors, where NNNNNNNNNN represents an IonXpress barcode); End-repair II using T4 DNA polymerase (NEB) and dNTP mix (NEB).

    Techniques: Ligation, Sequencing, Hybridization, Two Tailed Test

    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

    Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Journal: BMC Molecular Biology

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles

    doi: 10.1186/s12867-017-0083-2

    Figure Lengend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length

    Article Snippet: Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA).

    Techniques: Methylation, Ligation, Polymerase Chain Reaction, Amplification

    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