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    New England Biolabs t4 rna ligase 1
    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using <t>T4</t> RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.
    T4 Rna Ligase 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1875 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 rna ligase 1 - by Bioz Stars, 2020-05
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    99
    New England Biolabs t4 rnl1 ligase
    Experimental strategy towards mapping the 5′ and 3′ ends and analyzing the RNA editing status of plastid ndhD transcripts. In the upper panel, the location and orientation of primers for cDNA synthesis and PCR are shown relative to the ndhD coding region. Relevant restriction sites for cloning are indicated. Transcripts are self-ligated with <t>T4</t> RNA ligase, thereby fusing their 5′ and 3′ ends to produce circularized mRNA molecules. After cDNA synthesis primed with an ndhD -specific oligonucleotide, the region containing the 5′ UTR, 3′ UTR and the RNA editing site within the ndhD start codon is amplified by PCR. Products are then cloned and individual clones are sequenced to determine the termini of the mRNAs and the editing status of the start codon.
    T4 Rnl1 Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rnl1 ligase/product/New England Biolabs
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    t4 rnl1 ligase - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: ( a ) Schematic illustration of the high efficiency, purification- and template-free, adapter oligonucleotide adenylation method using T4 RNA ligase 1. The 3′ end of the adapter oligo was blocked by –ddC modification to prevent circularization and concatemerization. The 5′ base (shown in black) was swapped between dA, dC, dG, dT, rA, rC, rG, and rU to test bias. ( b ) The adapter adenylation efficiency was investigated as a function of 5′ terminal nucleotide. The reaction conditions were modified to exaggerate differences in efficiency (10 μL volume, 100 units ligase per nanomole adapter, 0.1 nanomole adapter, 30% PEG, 1 hour incubation). The rC and dG adapters are the most and least efficiently adenylated, respectively. ( c ) The adapter adenylation efficiency was then measured as a function of PEG % for a few representative adapters. In all cases, efficiency monotonically increased with PEG %. ( d ) Comparison of adenylation efficiency of as a function of PEG % under standard reaction conditions using the rA and dA adapters. Both the dA and rA adapters are efficiently adenylated at 35% PEG.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Purification, Modification, Incubation

    microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: microRNA-adapter ligation was performed using adenylated adapters generated by either (a) T4 RNA ligase 1 or (c) archaeal RNA ligase. The adapters were labeled with Cy5 while the synthetic microRNA were labeled with Cy3. Lanes 1 and 2 show that both methods are capable of fully adenylating the adapters. Lanes 4 and 6 show that let-7a microRNA can be effectively ligated both in the absence and presence of total RNA background. Lane 5 shows that large RNA molecules within the total RNA are captured by both adapters. No de-adenylation is observed with either method. ( b ) The T4 RNA ligase 1 adenylated adapter was used to capture RNA from 10, 100, or 1000 ng of pancreatic tissue total RNA spiked with 0.01 picomoles of 6 synthetic microRNA. The three ligation products from the top are large RNA molecules intrinsic to the total RNA that have been captured by the adapter. As expected, they vary in linear proportion to the total RNA input. The band in the middle is the spiked microRNA captured by the adapter which remains constant across all three samples as expected. The large band at the bottom of the gel is free adenylated Cy5 adapter.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Ligation, Generated, Labeling

    Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Journal: Scientific Reports

    Article Title: Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

    doi: 10.1038/srep15620

    Figure Lengend Snippet: Adenylated adapters generated using either T4 RNA ligase 1 or archaeal RNA ligase were used for microRNA-adapter ligation of a mixture containing 80 nt let-7a precursor DNA molecules and 22 nt let-7a mature microRNA molecules. The amount of PEG in the reaction mixture was also varied. Circularized DNA ligation product is only generated using the archaeal RNA ligase adenylated adapters.

    Article Snippet: Unless otherwise indicated, the adenylation reaction was performed using the optimized conditions of a 25 μL reaction volume containing 0.05 nanomole dA adapter, 1X T4 RNA Ligase Buffer (New England Biolabs, Ipswich, MA), 35% PEG, 1 mM ATP, and 300 units of T4 RNA Ligase 1 (New England Biolabs, Ipswich, MA) per nanomole adapter.

    Techniques: Generated, Ligation, DNA Ligation

    Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Journal: BMC Genomics

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    doi: 10.1186/s12864-017-3725-3

    Figure Lengend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Article Snippet: Cross-linked RNA molecules were then ligated using 40 U of T4 RNA ligase 1 (New England Biolabs, M0204), 1 mM ATP, and 40 U RNase inhibitors in RNA ligase 1 buffer for 1 h at 15 °C, and kept for 16 h at 4 °C.

    Techniques: Modification, Irradiation

    Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Journal: PLoS ONE

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    doi: 10.1371/journal.pone.0082176

    Figure Lengend Snippet: Identification of leaderless genomic RNA during BCoV persistent infection. (A) Strategy to identify positive-strand leaderless genomic RNA. Poly(A)-containing RNA was selected from total cellular RNA extracted from BCoV-persistently infected cells, treated with alkaline phosphatase, decapped with tobacco acid pyrophosphatase, head-to-tail ligated with T4 RNA ligase I, and used as the template for RT-PCR with the BCoV 5′ UTR-(+)-strand-specific primer 2: BCV107(+) (for RT) and BCoV 3′ UTR-(−)-strand-specific primer 1: BCV3′UTR1(−). (B) RT-PCR product synthesized by the method described in Fig. 1A. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) and with a size of less than 200 bp (lanes 6–7, marked with white arrowhead) were observed. (C) The upper panel shows part of the first 88-nt sequence of the 5′ UTR in the positive-strand BCoV genomic RNA. The positions (1 and 70) are given on the top of the sequence, and the intergenic sequence (IS) UCUAAAC is underlined. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of more than 200 bp from lane 7, as indicated with a black arrowhead in Fig. 1B. (D) The upper panel shows the sequence of the 5′UTR on the positive-strand BCoV genomic RNA, which lacks the first 69 nts; position 70 is given on the top of the sequence. The lower panel shows the sequence (shown in the negative strand) of the cDNA-cloned RT-PCR product with a size of less than 200 bp from lane 7, as indicated with a white arrowhead in Fig. 1B. (E) Control reactions to determine if the positive-strand leaderless genome is a degradation product. RT-PCR product was synthesized by the method described in Fig. 1A except RNA sample was not treated with alkaline phosphatase and tobacco acid pyrophosphatase. RT-PCR products with a length of more than 200 bp were detected. (F) Identification of negative-strand leaderless genomic RNA. Total cellular RNA was treated with tobacco acid pyrophosphatase and ligated with T4 RNA ligase I. RT-PCR product was synthesized by the method described in Fig. 1A except that primer BCV3′UTR1(−) was used for RT. RT-PCR products with a size of more than 200 bp (lanes 2–7, marked with black arrowhead) were observed. M, ds DNA size markers in nt pairs. dpi: days postinfection.

    Article Snippet: To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Clone Assay

    Experimental strategy towards mapping the 5′ and 3′ ends and analyzing the RNA editing status of plastid ndhD transcripts. In the upper panel, the location and orientation of primers for cDNA synthesis and PCR are shown relative to the ndhD coding region. Relevant restriction sites for cloning are indicated. Transcripts are self-ligated with T4 RNA ligase, thereby fusing their 5′ and 3′ ends to produce circularized mRNA molecules. After cDNA synthesis primed with an ndhD -specific oligonucleotide, the region containing the 5′ UTR, 3′ UTR and the RNA editing site within the ndhD start codon is amplified by PCR. Products are then cloned and individual clones are sequenced to determine the termini of the mRNAs and the editing status of the start codon.

    Journal: Nucleic Acids Research

    Article Title: Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon

    doi: 10.1093/nar/gkh217

    Figure Lengend Snippet: Experimental strategy towards mapping the 5′ and 3′ ends and analyzing the RNA editing status of plastid ndhD transcripts. In the upper panel, the location and orientation of primers for cDNA synthesis and PCR are shown relative to the ndhD coding region. Relevant restriction sites for cloning are indicated. Transcripts are self-ligated with T4 RNA ligase, thereby fusing their 5′ and 3′ ends to produce circularized mRNA molecules. After cDNA synthesis primed with an ndhD -specific oligonucleotide, the region containing the 5′ UTR, 3′ UTR and the RNA editing site within the ndhD start codon is amplified by PCR. Products are then cloned and individual clones are sequenced to determine the termini of the mRNAs and the editing status of the start codon.

    Article Snippet: A 2 µl aliquot of RNA from fractions P1 and P6 (EDTA-free polysome gradients) and 0.5 µl of RNA from fraction C10 (EDTA-containing polysome gradient) were ligated at 37°C for 1 h with 20 U of T4 RNA ligase (New England Biolabs) in a final volume of 100 µl. cDNA synthesis was performed as follows.

    Techniques: Polymerase Chain Reaction, Clone Assay, Amplification