ligation sequencing kit  (New England Biolabs)


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    Structured Review

    New England Biolabs ligation sequencing kit
    Ligation Sequencing Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ligation sequencing kit/product/New England Biolabs
    Average 96 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    ligation sequencing kit - by Bioz Stars, 2019-10
    96/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI
    Article Snippet: Paragraph title: Cloning ... The DNA sequence of most of the larger PstI fragment ( , ) was determined by a thermocycling procedure using the Circumvent Sequencing Kit (New England Biolabs).

    Centrifugation:

    Article Title: Circulating microRNAs and life expectancy among identical twins
    Article Snippet: After centrifugation, plasma aliquots were frozen at −70 °C ( ; ). .. The sequencing libraries were constructed using the New England Biolabs (NEB) small RNA sequencing kit and were analyzed on an Illumina HiSeq 2000 platform with single end 50 nucleotide read length.

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: After fragmentation, the immunoprecipitated complexes were purified via centrifugation at 5,000xg for 2 minutes and washed three times with RIPA buffer and twice more with sterile 1xPBS. .. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions.

    Article Title: A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics
    Article Snippet: The mixture was pelleted by centrifugation (5,000 × g for 5 min) and resuspended in 1 ml LB broth. .. Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB).

    Amplification:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: In order to generate sufficient amount of cDNAs for MinION library preparation, samples were amplified by endpoint PCR following the TeloPrime Kit’s manual. .. The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends.

    Article Title: The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils
    Article Snippet: Amplification products were purified with the QIAquick PCR purification kit (Qiagen), and pooled in roughly equimolar quantities. .. Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set). .. Index tagged samples were amplified (8 cycles of PCR, KAPA HiFi kit, KAPA Biosystems), quantified by qPCR (KAPA Library Quant Kit, KAPA Biosystems) and submitted to cluster formation for MiSeq sequencing (300 BP PE read length, Illumina).

    Synthesized:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: Briefly, the DNA was end-repaired and A-tailed using NEBNext ChIP-Seq Library Prep Master Mix (NEB, E6240 kit) as described in the kit’s protocol. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP . .. Briefly, 100 ng of al-DNA was diluted in 42 μl of TE buffer.

    Methylated DNA Immunoprecipitation Sequencing:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: Paragraph title: MeDIP-seq details ... Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP .

    Autoradiography:

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs). .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs).

    Construct:

    Article Title: Circulating microRNAs and life expectancy among identical twins
    Article Snippet: We performed miRNA profiling using NGS at the Vanderbilt Center for Genomic Research. .. The sequencing libraries were constructed using the New England Biolabs (NEB) small RNA sequencing kit and were analyzed on an Illumina HiSeq 2000 platform with single end 50 nucleotide read length. .. Post sequencing data analysis was performed with an in-house small RNA sequence analysis pipeline as described ( ).

    Article Title: Capture and Amplification by Tailing and Switching (CATS)
    Article Snippet: As a result, besides their time and labor intensiveness, adaptor-ligation methods demand additional purification steps before pre-amplification of the cDNA libraries and have a limited number of possible pre-amplification cycles. .. To construct DNA libraries suitable for Illumina MiSeq or HiSeq platforms we have used adaptor sequences from the NEBnext Small RNA Sequencing Kit (New England Biolabs). .. The sequence corresponding to the 5′-adaptor was incorporated into the TSO and the 3′-adaptor sequences was used to design a terminal tag of the poly(dT) primer ( ).

    Incubation:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: The pellet was carefully washed once with 70% Ethanol and resuspended in 100 μl of TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) with 20 μgml−1 RNase A and incubated for 30 min at 37 °C followed by 1 h at 65 °C. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP .

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: After the antibody binding period, the immunocomplexes were captured from the lysate by the addition of 100 μl (packed volume) of protein G Sepharose resin and an additional 2-h incubation period at 4°C under agitation. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions.

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: Primer extension buffer was added (final concentration, 50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol) with 10,000 U of Superscript AMVRT (Gibco BRL). .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs). .. These sequencing ladders were denatured as above and loaded next to the primer extension products for visualization of the transcriptional start site.

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
    Article Snippet: Purified polyadenylated RNA was mixed with 2.5 μg of affinity purified anti-m6 A polyclonal antibody (202003; Synaptic Systems, Goettingen, Germany) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) and incubated for 2 hr at 4°C. .. RNA was used for library generation with the small RNA sequencing kit (NEB, Ipswich, MA).

    Article Title: A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics
    Article Snippet: Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB). .. Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB).

    In Silico:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends. .. This step was followed by the 1D adapter ligation, which was carried out according to the 1D protocol, using the NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Transformation Assay:

    Article Title: Circulating microRNAs and life expectancy among identical twins
    Article Snippet: The sequencing libraries were constructed using the New England Biolabs (NEB) small RNA sequencing kit and were analyzed on an Illumina HiSeq 2000 platform with single end 50 nucleotide read length. .. Post sequencing data analysis was performed with an in-house small RNA sequence analysis pipeline as described ( ).

    Derivative Assay:

    Article Title: The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils
    Article Snippet: The SNVs selected for validation were derived from computation sets found in both DFT1s (86T and 88T), both DFT2s (202T2 and 203T3) or in all four tumors (86T, 88T, 202T2, 203T3). .. Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set).

    Hybridization:

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: The RNA was then resuspended in 12.5 μl of hybridization buffer (final concentration, 150 mM KCl, 10 mM Tris-HCl [pH 8.3], 1 mM EDTA); 0.8 pmol of labeled probe was added and allowed to anneal for 30 min at 65°C after being denatured for 5 min at 95°C. .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs).

    Flow Cytometry:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends. .. The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends.

    Ligation:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The generation of the sequencing-ready library from this sample is based on the 1D strand switching cDNA by ligation protocol from ONT. .. The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends. .. This step was followed by the 1D adapter ligation, which was carried out according to the 1D protocol, using the NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Transferring:

    Article Title: A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics
    Article Snippet: After 3 h of incubation, cells were scraped off the filter disks using a pipette tip into LB medium containing streptomycin, vortexed, and plated directly onto LB agar plates containing streptomycin (to kill the donor strain) and either no antibiotic (control library), 200 μg/ml vancomycin, or 200 μg/ml bacitracin. .. Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB).

    Infection:

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions. .. The specificity of the immunoprecipitation protocol described above was confirmed independently using small scale analytical replicates and metabolic labeling.

    Polymerase Chain Reaction:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: In order to generate sufficient amount of cDNAs for MinION library preparation, samples were amplified by endpoint PCR following the TeloPrime Kit’s manual. .. The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends.

    Article Title: The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils
    Article Snippet: Amplification products were purified with the QIAquick PCR purification kit (Qiagen), and pooled in roughly equimolar quantities. .. Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set).

    Article Title: Capture and Amplification by Tailing and Switching (CATS)
    Article Snippet: To construct DNA libraries suitable for Illumina MiSeq or HiSeq platforms we have used adaptor sequences from the NEBnext Small RNA Sequencing Kit (New England Biolabs). .. To construct DNA libraries suitable for Illumina MiSeq or HiSeq platforms we have used adaptor sequences from the NEBnext Small RNA Sequencing Kit (New England Biolabs).

    Sonication:

    Article Title: A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics
    Article Snippet: Libraries for Illumina sequencing were prepared as described previously ( ). .. Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB). .. This was followed by Illumina adaptor ligation and amplification of genomic DNA-transposon junctions.

    Affinity Purification:

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
    Article Snippet: Purified polyadenylated RNA was mixed with 2.5 μg of affinity purified anti-m6 A polyclonal antibody (202003; Synaptic Systems, Goettingen, Germany) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) and incubated for 2 hr at 4°C. .. RNA was used for library generation with the small RNA sequencing kit (NEB, Ipswich, MA).

    Binding Assay:

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: After binding, the RNAs present in the lysate slurry were fragmented by the addition of RNase T1 to each immunoprecipitation. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions.

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: After resin binding the RNAs were fragmented via the addition of RNAse T1 (ThermoScientific) to each immunoprecipitation. .. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions.

    ChIP-sequencing:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: Briefly, the DNA was end-repaired and A-tailed using NEBNext ChIP-Seq Library Prep Master Mix (NEB, E6240 kit) as described in the kit’s protocol. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP .

    Article Title: PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus by preventing de novo DNA methylation in mouse embryonic stem cells
    Article Snippet: Paragraph title: ChIP sequencing (ChIP-Seq) and Library Generation ... NEB next generation sequencing kit was used to prepare the libraries.

    DNA Extraction:

    Article Title: A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics
    Article Snippet: Libraries for Illumina sequencing were prepared as described previously ( ). .. Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB). .. This was followed by Illumina adaptor ligation and amplification of genomic DNA-transposon junctions.

    RNA Sequencing Assay:

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: The RNA components of the purified RNA-protein complexes were released by proteinase K digestion at 37°C in the presence of 1.0% SDS. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions. .. The resulting cDNA libraries were sequenced using the Illumina MiSeq platform to yield cDNA libraries by next-generation sequencing.

    Article Title: Circulating microRNAs and life expectancy among identical twins
    Article Snippet: We performed miRNA profiling using NGS at the Vanderbilt Center for Genomic Research. .. The sequencing libraries were constructed using the New England Biolabs (NEB) small RNA sequencing kit and were analyzed on an Illumina HiSeq 2000 platform with single end 50 nucleotide read length. .. Post sequencing data analysis was performed with an in-house small RNA sequence analysis pipeline as described ( ).

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
    Article Snippet: Purified polyadenylated RNA was mixed with 2.5 μg of affinity purified anti-m6 A polyclonal antibody (202003; Synaptic Systems, Goettingen, Germany) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) and incubated for 2 hr at 4°C. .. RNA was used for library generation with the small RNA sequencing kit (NEB, Ipswich, MA). .. Sequencing was carried out on Illumina HiSeq 2000 according to the manufacturer’s instructions.

    Article Title: The extent of ribosome queuing in budding yeast
    Article Snippet: Ribosome profiling was performed as previously described [ , ], with minor modifications. rRNA was depleted using Ribo-Zero by EpiCentre. .. The small RNA sequencing kit from New England BioLabs [ ] was utilized. .. Sucrose gradient fractionation was employed for filtering reads containing one or two ribosomes.

    Article Title: Capture and Amplification by Tailing and Switching (CATS)
    Article Snippet: As a result, besides their time and labor intensiveness, adaptor-ligation methods demand additional purification steps before pre-amplification of the cDNA libraries and have a limited number of possible pre-amplification cycles. .. To construct DNA libraries suitable for Illumina MiSeq or HiSeq platforms we have used adaptor sequences from the NEBnext Small RNA Sequencing Kit (New England Biolabs). .. The sequence corresponding to the 5′-adaptor was incorporated into the TSO and the 3′-adaptor sequences was used to design a terminal tag of the poly(dT) primer ( ).

    Transfection:

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: Total RNA was collected from cells transfected as above by the guanidinium isothiocyanate method ( , ) or by passage through an RNeasy spin column (Qiagen, Valencia, Calif.). .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs).

    Labeling:

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: The RNA was then resuspended in 12.5 μl of hybridization buffer (final concentration, 150 mM KCl, 10 mM Tris-HCl [pH 8.3], 1 mM EDTA); 0.8 pmol of labeled probe was added and allowed to anneal for 30 min at 65°C after being denatured for 5 min at 95°C. .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs).

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions. .. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions.

    Purification:

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: The RNA components of the purified RNA-protein complexes were released by proteinase K digestion at 37°C in the presence of 1.0% SDS. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions. .. The resulting cDNA libraries were sequenced using the Illumina MiSeq platform to yield cDNA libraries by next-generation sequencing.

    Article Title: PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus by preventing de novo DNA methylation in mouse embryonic stem cells
    Article Snippet: Purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. .. NEB next generation sequencing kit was used to prepare the libraries.

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
    Article Snippet: Purified polyadenylated RNA was mixed with 2.5 μg of affinity purified anti-m6 A polyclonal antibody (202003; Synaptic Systems, Goettingen, Germany) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.4) and incubated for 2 hr at 4°C. .. RNA was used for library generation with the small RNA sequencing kit (NEB, Ipswich, MA).

    Article Title: The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils
    Article Snippet: Amplification products were purified with the QIAquick PCR purification kit (Qiagen), and pooled in roughly equimolar quantities. .. Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set). .. Index tagged samples were amplified (8 cycles of PCR, KAPA HiFi kit, KAPA Biosystems), quantified by qPCR (KAPA Library Quant Kit, KAPA Biosystems) and submitted to cluster formation for MiSeq sequencing (300 BP PE read length, Illumina).

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: The purified RNA fragments were then extracted using TRIzol and resuspended in a minimal volume. .. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions.

    Article Title: Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cells identity
    Article Snippet: Purified ChIP DNA was used to prepare illumina multiplexed sequencing libraries. .. NEB next generation sequencing kit was used to prepare the libraries.

    Sequencing:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: The generation of the sequencing-ready library from this sample is based on the 1D strand switching cDNA by ligation protocol from ONT. .. The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends. .. This step was followed by the 1D adapter ligation, which was carried out according to the 1D protocol, using the NEB Blunt/TA Ligase Master Mix (New England Biolabs).

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: Briefly, the DNA was end-repaired and A-tailed using NEBNext ChIP-Seq Library Prep Master Mix (NEB, E6240 kit) as described in the kit’s protocol. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP . .. Briefly, 100 ng of al-DNA was diluted in 42 μl of TE buffer.

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions.

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: Primer extension buffer was added (final concentration, 50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol) with 10,000 U of Superscript AMVRT (Gibco BRL). .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs). .. These sequencing ladders were denatured as above and loaded next to the primer extension products for visualization of the transcriptional start site.

    Article Title: PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus by preventing de novo DNA methylation in mouse embryonic stem cells
    Article Snippet: Paragraph title: ChIP sequencing (ChIP-Seq) and Library Generation ... NEB next generation sequencing kit was used to prepare the libraries.

    Article Title: Circulating microRNAs and life expectancy among identical twins
    Article Snippet: We performed miRNA profiling using NGS at the Vanderbilt Center for Genomic Research. .. The sequencing libraries were constructed using the New England Biolabs (NEB) small RNA sequencing kit and were analyzed on an Illumina HiSeq 2000 platform with single end 50 nucleotide read length. .. Post sequencing data analysis was performed with an in-house small RNA sequence analysis pipeline as described ( ).

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
    Article Snippet: High-throughput sequencing of HIV-1 methylome was carried out using m6 A-seq ( ) and followed the protocol published previously ( ). .. RNA was used for library generation with the small RNA sequencing kit (NEB, Ipswich, MA).

    Article Title: The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils
    Article Snippet: Amplification products were purified with the QIAquick PCR purification kit (Qiagen), and pooled in roughly equimolar quantities. .. Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set). .. Index tagged samples were amplified (8 cycles of PCR, KAPA HiFi kit, KAPA Biosystems), quantified by qPCR (KAPA Library Quant Kit, KAPA Biosystems) and submitted to cluster formation for MiSeq sequencing (300 BP PE read length, Illumina).

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: The purified RNA fragments were then extracted using TRIzol and resuspended in a minimal volume. .. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions. .. The resulting cDNA libraries were sequenced using the MiSeq platform.

    Article Title: The extent of ribosome queuing in budding yeast
    Article Snippet: The small RNA sequencing kit from New England BioLabs [ ] was utilized. .. The small RNA sequencing kit from New England BioLabs [ ] was utilized.

    Article Title: Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cells identity
    Article Snippet: Paragraph title: ChIP sequencing and Library Generation ... NEB next generation sequencing kit was used to prepare the libraries.

    Article Title: Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI
    Article Snippet: The genes for the HinP1I R-M system were cloned into Escherichia coli by the ‘methylase-selection’ technique ( , ). .. The DNA sequence of most of the larger PstI fragment ( , ) was determined by a thermocycling procedure using the Circumvent Sequencing Kit (New England Biolabs). .. The primers, 14–16 bp in length and spaced at intervals of ∼250 nt, were obtained from New England Biolabs.

    Article Title: A Transposon Screen Identifies Genetic Determinants of Vibrio cholerae Resistance to High-Molecular-Weight Antibiotics
    Article Snippet: Paragraph title: Transposon insertion sequencing. ... Briefly, following genomic DNA extraction, DNA was sheared by using sonication, and overhanging ends were blunted by using a blunting enzyme kit (NEB).

    Immunoprecipitation:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: Briefly, the DNA was end-repaired and A-tailed using NEBNext ChIP-Seq Library Prep Master Mix (NEB, E6240 kit) as described in the kit’s protocol. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP . .. Briefly, 100 ng of al-DNA was diluted in 42 μl of TE buffer.

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: After binding, the RNAs present in the lysate slurry were fragmented by the addition of RNase T1 to each immunoprecipitation. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions.

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: Paragraph title: Cross-linked immunoprecipitation–deep sequencing (CLIP-seq) of SINV C:R complexes ... The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions.

    cDNA Library Assay:

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: The RNA components of the purified RNA-protein complexes were released by proteinase K digestion at 37°C in the presence of 1.0% SDS. .. The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions. .. The resulting cDNA libraries were sequenced using the Illumina MiSeq platform to yield cDNA libraries by next-generation sequencing.

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: The purified RNA fragments were then extracted using TRIzol and resuspended in a minimal volume. .. The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions. .. The resulting cDNA libraries were sequenced using the MiSeq platform.

    Chromatin Immunoprecipitation:

    Article Title: PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus by preventing de novo DNA methylation in mouse embryonic stem cells
    Article Snippet: Paragraph title: ChIP sequencing (ChIP-Seq) and Library Generation ... NEB next generation sequencing kit was used to prepare the libraries.

    Article Title: Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cells identity
    Article Snippet: Paragraph title: ChIP sequencing and Library Generation ... NEB next generation sequencing kit was used to prepare the libraries.

    Plasmid Preparation:

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: C6/36 cells were transfected as above except that 75-cm2 flasks seeded with 2.25 × 107 cells were incubated with 800 μl of transfection mixture (150 μl of Lipofectin and 30 μg of plasmid DNA in L15). .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs).

    Selection:

    Article Title: Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus
    Article Snippet: Paragraph title: Cap selection, cDNA synthesis and sequencing ... The Ligation Sequencing kit (SQK-LSK108, ONT) and NEBNext End repair / dA-tailing Module (New England Biolabs) was used to repair the cDNA ends.

    Next-Generation Sequencing:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: The genomic DNA was sheared using a Bioruptor NGS (Diagenode) for 10 cycles (30 s ON, 30 s OFF) to an average size of 250–300 bp and ~700 ng of the sheared DNA was used further for library preparation. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP .

    Article Title: PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus by preventing de novo DNA methylation in mouse embryonic stem cells
    Article Snippet: Purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. .. NEB next generation sequencing kit was used to prepare the libraries. .. All ChIP-Seq samples were aligned with Bowtie v0.12.9 to the mm9 genome assembly, where only uniquely mappable reads were reported.

    Article Title: Circulating microRNAs and life expectancy among identical twins
    Article Snippet: Paragraph title: miRNA profiling using next-generation sequencing (NGS) ... The sequencing libraries were constructed using the New England Biolabs (NEB) small RNA sequencing kit and were analyzed on an Illumina HiSeq 2000 platform with single end 50 nucleotide read length.

    Article Title: Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cells identity
    Article Snippet: Purified ChIP DNA was used to prepare illumina multiplexed sequencing libraries. .. NEB next generation sequencing kit was used to prepare the libraries. .. Peaks were called using Model-based Analysis for ChIP-Seq (MACS), peak-finding algorithm to identify regions of ChIP-seq enrichment over background (Zhang et al., 2008).

    Concentration Assay:

    Article Title: Characterization of the Structural Gene Promoter of Aedes aegypti Densovirus
    Article Snippet: Primer extension buffer was added (final concentration, 50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol) with 10,000 U of Superscript AMVRT (Gibco BRL). .. The reaction mixture was incubated at 42°C for 50 min, and the reaction was stopped by heating to 70°C for 10 min. Then 4 μl of gel loading/stop buffer was added (New England Biolabs), and the samples were denatured at 95°C for 10 min and separated on a 5% acrylamide–8 M urea sequencing gel at 1,500 V for 2.5 h. To determine the precise transcriptional start site, sequencing was performed on nsp61gal or pUCA as a template with the same oligonucleotides as used for primer extension, using a Circumvent sequencing kit (New England Biolabs).

    Methylated DNA Immunoprecipitation:

    Article Title: Genome-wide chromatin and gene expression profiling during memory formation and maintenance in adult mice
    Article Snippet: Briefly, the DNA was end-repaired and A-tailed using NEBNext ChIP-Seq Library Prep Master Mix (NEB, E6240 kit) as described in the kit’s protocol. .. Custom synthesized Illumina paired-end sequencing adaptors (Sigma Aldrich) were ligated as mentioned in the NEB-E6240 kit protocol and 100 ng of the adapter ligated DNA fragments (al-DNA) was used for immunoprecipitation using anti-5-methylcytosine (5 mC) antibody as described earlier for MeDIP . .. Briefly, 100 ng of al-DNA was diluted in 42 μl of TE buffer.

    High Throughput Screening Assay:

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression
    Article Snippet: High-throughput sequencing of HIV-1 methylome was carried out using m6 A-seq ( ) and followed the protocol published previously ( ). .. RNA was used for library generation with the small RNA sequencing kit (NEB, Ipswich, MA).

    Variant Assay:

    Article Title: The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils
    Article Snippet: Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set). .. Pooled amplicon DNA was quantified (dsDNA BR assay, Thermo Fisher Scientific), purified, libraries prepared (NEBNext Sanger Sequencing Kit, New England Biolabs, Ipswich, MA, USA), and index tags applied (Sanger 168 tag set).

    Cross-linking Immunoprecipitation:

    Article Title: Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
    Article Snippet: Paragraph title: CLIP-seq identification of hnRNP interaction sites. ... The released RNA fragments were TRIzol extracted, and the purified RNA fragments were then used as the input material for cDNA library generation with the NEBNext small RNA sequencing kit, according to the manufacturer's directions.

    Article Title: Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants
    Article Snippet: Paragraph title: Cross-linked immunoprecipitation–deep sequencing (CLIP-seq) of SINV C:R complexes ... The RNA fragments were then used as the input materials for cDNA library generation via the NEBNext sequencing kit, as according to the manufacturer’s instructions.

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    New England Biolabs nebuilder hifi assembly
    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by <t>NEBuilder</t> <t>HiFi</t> assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Nebuilder Hifi Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ultra directional rna library prep kit
    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on <t>RNA</t> purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S <t>rRNA</t> (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.
    Ultra Directional Rna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna cleanup kit
    Frequency of specific base pair mismatches by position. Incidence of each possible mismatched base pair observed for ligation of three-base overhangs. The results shown are for SMRT sequencing of ligation reactions with 100 nM of the multiplexed three-base overhang substrate, 1 h at 25°C ( A and B ) or <t>37°C</t> ( C and D ), with 1.75 μM T4 <t>DNA</t> ligase in standard ligation buffer. This figure was generated from the same data as shown in Figures 2 and 3 . A and C show the results for the edge position (N1:N3′); B and D for the middle position (N2:N2′).
    Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [ ], uracil-specific excision reagent cloning (USER) [ ], Gibson assembly [ ], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [ ], transformation-associated recombination (TAR) cloning [ ] and seamless ligation cloning extract (SLiCE) [ ], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [ ]).

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Article Snippet: In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [ ], uracil-specific excision reagent cloning (USER) [ ], Gibson assembly [ ], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [ ], transformation-associated recombination (TAR) cloning [ ] and seamless ligation cloning extract (SLiCE) [ ], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [ ]).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: In contrast to Golden Gate and derived methods, overlap-depended assembly methods, such as circular polymerase extension cloning (CPEC) [ ], uracil-specific excision reagent cloning (USER) [ ], Gibson assembly [ ], NeBuilder HiFi assembly (NEB), sequence and ligation independent cloning (SLIC) [ ], transformation-associated recombination (TAR) cloning [ ] and seamless ligation cloning extract (SLiCE) [ ], are sequence independent and therefore convenient and efficient when performing multigene cloning (see review [ ]).

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Liquid Chromatography, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Article Snippet: RNA sample preparation (total RNA and polysomal RNA) from E. coli strain MC4100 F΄ without or with plasmid pSA1 15 min upon induction of mazF expression was described elsewhere ( ). cDNA libraries were prepared using 50–100 ng of the rRNA-depleted RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), following the manufacturer's instructions and sequenced on Illumina HiSequ2000 (read length 100bp).

    Techniques: Purification, In Vitro, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Ligation, Sequencing

    RNA43, 16S Δ43 rRNA and 70S Δ43 ribosomes are stable during stress conditions. ( A ) Schematic depiction of the experimental approach to assess the stability of RNA43 and 16S Δ43 rRNA in vivo . A schematic growth curve in the absence of IPTG is shown in black. At OD 600 of 0.3 the culture was divided and IPTG was added to one half to induce mazF expression (growth is blocked as indicated in red). 60 minutes thereafter, cells were washed and resuspended in fresh medium comprising rifampicin (in blue). ( B ) Samples withdrawn at the time points indicated were subjected to northern blot analysis with probes specific for the central domain of the 16S rRNA (CD, panel a), the 3΄-terminus of the 16S rRNA (A20; panel b) and the RNA43 (A20; panel c). 5S rRNA was used as internal standard for quantification (panel d). In vitro transcribed 16S rRNA (lane 1) and RNA43 (lane 2) served as size markers. The experiment was performed in triplicate and one representative autoradiograph is shown. ( C ) Schematic of the experimental approach to assess the stability of 70S Δ43 ribosomes in vivo as described in A. ( D ) S30 extracts were prepared before (dotted lines) and 120 min after addition of rifampicin (solid lines) to untreated cells (black lines) or 60 min after induction of mazF expression (red lines) and subjected to sucrose density gradient analysis. Peaks representing 30S and 50S subunits and 70S ribosomes are indicated. The peak areas of the 30S and 50S subunits (filled areas) and 70S monosomes (hatched area) that were quantified to determine the subunits/monosome ratios are indicated. ( E ) To monitor MazF-mediated processing of the 16S rRNA, RNA was isolated from the fractions comprising the 70S monosomes. RT-PCR analysis using primers S7/X15 (upper panel), specific for both intact 16S rRNA and 16S Δ43 rRNA, and primers S7/Y12 (lower panel), yielding a product only with uncleaved 16S rRNA. NTC: no template control. Below, the binding sites of the primers are given schematically. ( F ) Ribosome sedimentation profiles of cell extracts 30 min after induction of mazF expression. Total RNA was purified from the indicated fractions (top, 30S, 50S, 70S and polysomes), respectively, and tested for the presence of RNA43 by northern blotting. Total RNA purified from the S30 extract withdrawn 30 min upon induction of mazF expression (lane 1), and in vitro transcribed RNA43 (lane 7) served as controls.

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: RNA43, 16S Δ43 rRNA and 70S Δ43 ribosomes are stable during stress conditions. ( A ) Schematic depiction of the experimental approach to assess the stability of RNA43 and 16S Δ43 rRNA in vivo . A schematic growth curve in the absence of IPTG is shown in black. At OD 600 of 0.3 the culture was divided and IPTG was added to one half to induce mazF expression (growth is blocked as indicated in red). 60 minutes thereafter, cells were washed and resuspended in fresh medium comprising rifampicin (in blue). ( B ) Samples withdrawn at the time points indicated were subjected to northern blot analysis with probes specific for the central domain of the 16S rRNA (CD, panel a), the 3΄-terminus of the 16S rRNA (A20; panel b) and the RNA43 (A20; panel c). 5S rRNA was used as internal standard for quantification (panel d). In vitro transcribed 16S rRNA (lane 1) and RNA43 (lane 2) served as size markers. The experiment was performed in triplicate and one representative autoradiograph is shown. ( C ) Schematic of the experimental approach to assess the stability of 70S Δ43 ribosomes in vivo as described in A. ( D ) S30 extracts were prepared before (dotted lines) and 120 min after addition of rifampicin (solid lines) to untreated cells (black lines) or 60 min after induction of mazF expression (red lines) and subjected to sucrose density gradient analysis. Peaks representing 30S and 50S subunits and 70S ribosomes are indicated. The peak areas of the 30S and 50S subunits (filled areas) and 70S monosomes (hatched area) that were quantified to determine the subunits/monosome ratios are indicated. ( E ) To monitor MazF-mediated processing of the 16S rRNA, RNA was isolated from the fractions comprising the 70S monosomes. RT-PCR analysis using primers S7/X15 (upper panel), specific for both intact 16S rRNA and 16S Δ43 rRNA, and primers S7/Y12 (lower panel), yielding a product only with uncleaved 16S rRNA. NTC: no template control. Below, the binding sites of the primers are given schematically. ( F ) Ribosome sedimentation profiles of cell extracts 30 min after induction of mazF expression. Total RNA was purified from the indicated fractions (top, 30S, 50S, 70S and polysomes), respectively, and tested for the presence of RNA43 by northern blotting. Total RNA purified from the S30 extract withdrawn 30 min upon induction of mazF expression (lane 1), and in vitro transcribed RNA43 (lane 7) served as controls.

    Article Snippet: RNA sample preparation (total RNA and polysomal RNA) from E. coli strain MC4100 F΄ without or with plasmid pSA1 15 min upon induction of mazF expression was described elsewhere ( ). cDNA libraries were prepared using 50–100 ng of the rRNA-depleted RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), following the manufacturer's instructions and sequenced on Illumina HiSequ2000 (read length 100bp).

    Techniques: In Vivo, Expressing, Northern Blot, In Vitro, Autoradiography, Isolation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Sedimentation, Purification

    Model for the reversible stress adaptation of the translational machinery in E. coli . When E. coli encounters stress (red) the endoribonuclease MazF is activated and removes the 3΄-terminal 43 nts (RNA43) from the 16S rRNA incorporated in active ribosomes (i). The resulting 70S Δ43 ribosomes selectively translate MazF-processed mRNAs to adapt protein synthesis to the adverse conditions (ii). Upon stress relief when canonical mRNAs are transcribed again, the specialized ribosomes become redundant. To regenerate these ribosomes the RNA ligase RtcB re-ligates the 16S Δ43 rRNA present in the stress-ribosomes and RNA43 (iii), thereby restoring the translational proficiency of 70S ribosomes to ensure canonical translation during relaxed conditions (green) (iv).

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: Model for the reversible stress adaptation of the translational machinery in E. coli . When E. coli encounters stress (red) the endoribonuclease MazF is activated and removes the 3΄-terminal 43 nts (RNA43) from the 16S rRNA incorporated in active ribosomes (i). The resulting 70S Δ43 ribosomes selectively translate MazF-processed mRNAs to adapt protein synthesis to the adverse conditions (ii). Upon stress relief when canonical mRNAs are transcribed again, the specialized ribosomes become redundant. To regenerate these ribosomes the RNA ligase RtcB re-ligates the 16S Δ43 rRNA present in the stress-ribosomes and RNA43 (iii), thereby restoring the translational proficiency of 70S ribosomes to ensure canonical translation during relaxed conditions (green) (iv).

    Article Snippet: RNA sample preparation (total RNA and polysomal RNA) from E. coli strain MC4100 F΄ without or with plasmid pSA1 15 min upon induction of mazF expression was described elsewhere ( ). cDNA libraries were prepared using 50–100 ng of the rRNA-depleted RNA with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7420), following the manufacturer's instructions and sequenced on Illumina HiSequ2000 (read length 100bp).

    Techniques:

    Frequency of specific base pair mismatches by position. Incidence of each possible mismatched base pair observed for ligation of three-base overhangs. The results shown are for SMRT sequencing of ligation reactions with 100 nM of the multiplexed three-base overhang substrate, 1 h at 25°C ( A and B ) or 37°C ( C and D ), with 1.75 μM T4 DNA ligase in standard ligation buffer. This figure was generated from the same data as shown in Figures 2 and 3 . A and C show the results for the edge position (N1:N3′); B and D for the middle position (N2:N2′).

    Journal: Nucleic Acids Research

    Article Title: A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining

    doi: 10.1093/nar/gky303

    Figure Lengend Snippet: Frequency of specific base pair mismatches by position. Incidence of each possible mismatched base pair observed for ligation of three-base overhangs. The results shown are for SMRT sequencing of ligation reactions with 100 nM of the multiplexed three-base overhang substrate, 1 h at 25°C ( A and B ) or 37°C ( C and D ), with 1.75 μM T4 DNA ligase in standard ligation buffer. This figure was generated from the same data as shown in Figures 2 and 3 . A and C show the results for the edge position (N1:N3′); B and D for the middle position (N2:N2′).

    Article Snippet: Reactions were halted by addition of 1 μl Proteinase K followed by 20 min incubation at 37°C, then purified using the Monarch® PCR & DNA Cleanup Kit (NEB).

    Techniques: Ligation, Sequencing, Generated

    Assay results for the ligation of randomized three-base overhangs by T4 DNA ligase (1 h at 37°C). SMRT sequencing results for ligating 100 nM of the multiplexed three-base overhang substrate 1 h at 37°C, with 1.75 μM T4 DNA ligase in standard ligation buffer. Observations have been normalized to 100 000 ligation events (see Supplementary Data for actual observation totals). ( A ) Frequency heat map of all ligation events (log-scaled). Overhangs are listed alphabetically left to right (AAA, AAC, AAG …TTG, TTT) and bottom to top such that the Watson–Crick pairings are shown on the diagonal. ( B ) Stacked bar plot showing the frequency of ligation products containing each overhang, corresponding to each column in the heat map in (A). Fully Watson–Crick paired ligation results are indicated in blue, and ligation products containing one or more mismatches are in orange.

    Journal: Nucleic Acids Research

    Article Title: A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining

    doi: 10.1093/nar/gky303

    Figure Lengend Snippet: Assay results for the ligation of randomized three-base overhangs by T4 DNA ligase (1 h at 37°C). SMRT sequencing results for ligating 100 nM of the multiplexed three-base overhang substrate 1 h at 37°C, with 1.75 μM T4 DNA ligase in standard ligation buffer. Observations have been normalized to 100 000 ligation events (see Supplementary Data for actual observation totals). ( A ) Frequency heat map of all ligation events (log-scaled). Overhangs are listed alphabetically left to right (AAA, AAC, AAG …TTG, TTT) and bottom to top such that the Watson–Crick pairings are shown on the diagonal. ( B ) Stacked bar plot showing the frequency of ligation products containing each overhang, corresponding to each column in the heat map in (A). Fully Watson–Crick paired ligation results are indicated in blue, and ligation products containing one or more mismatches are in orange.

    Article Snippet: Reactions were halted by addition of 1 μl Proteinase K followed by 20 min incubation at 37°C, then purified using the Monarch® PCR & DNA Cleanup Kit (NEB).

    Techniques: Ligation, Sequencing