t4 rna ligase  (New England Biolabs)


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  • 99
    Name:
    T4 RNA Ligase 2
    Description:
    T4 RNA Ligase 2 750 units
    Catalog Number:
    m0239l
    Price:
    312
    Size:
    750 units
    Category:
    RNA Ligases
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    Structured Review

    New England Biolabs t4 rna ligase
    T4 RNA Ligase 2
    T4 RNA Ligase 2 750 units
    https://www.bioz.com/result/t4 rna ligase/product/New England Biolabs
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus"

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.
    Figure Legend Snippet: The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Techniques Used: Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Sequencing

    2) Product Images from "Apoptotic signals induce specific degradation of ribosomal RNA in yeast"

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1100

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.
    Figure Legend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Techniques Used: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay

    3) Product Images from "A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage"

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq756

    RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.
    Figure Legend Snippet: RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Techniques Used: High Performance Liquid Chromatography, Purification, Expressing, Polyacrylamide Gel Electrophoresis, Ligation, Labeling

    4) Product Images from "A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage"

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1265

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .
    Figure Legend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Techniques Used: Ligation

    5) Product Images from "Human tRNA-derived small RNAs in the global regulation of RNA silencing"

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing

    Journal: RNA

    doi: 10.1261/rna.2000810

    Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except
    Figure Legend Snippet: Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except

    Techniques Used: Northern Blot, Derivative Assay

    6) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    7) Product Images from "Apoptotic signals induce specific degradation of ribosomal RNA in yeast"

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1100

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.
    Figure Legend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Techniques Used: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay

    8) Product Images from "A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage"

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1265

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .
    Figure Legend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Techniques Used: Ligation

    9) Product Images from "Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis"

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    Journal: Scientific Reports

    doi: 10.1038/srep28791

    Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.
    Figure Legend Snippet: Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.

    Techniques Used: Amplification, Isolation, Transgenic Assay, Purification, Polymerase Chain Reaction, Molecular Weight, Marker, Over Expression

    10) Product Images from "Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element"

    Article Title: Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

    Journal: RNA

    doi: 10.1261/rna.506607

    Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.
    Figure Legend Snippet: Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.

    Techniques Used: Labeling, Incubation

    The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.
    Figure Legend Snippet: The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.

    Techniques Used: Purification, Labeling, Ligation, Incubation

    11) Product Images from "Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease"

    Article Title: Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp537

    Splicing activity and specificity of the NEQ RNA splicing endonuclease. ( A ) A 3′-radiolabeled RNA transcript of the N. equitans tRNA Glu precursor was incubated without enzyme (–) or with either 1 μM NEQ205-NEQ261 splicing endonuclease at 65°C for 20 min alone or followed by incubation with T4 polynucleotide kinase (PNK) and T4 RNA ligase. AFU splicing endonuclease was incubated with a substrate as a positive control. ( B ) Secondary structure of the relaxed BHB motif of N. equitans tRNA Glu precursor. The predicted cleavage sites are indicated by arrows and the CUC anticodon is indicated by a line. ( C ) The mature tRNA product was excised, amplified by RT-PCR and sequenced. The anticodon loop was correctly assembled and the anticodon is underlined. ( D ) Examples of the RNA substrates cleaved by the tRNA splicing endonuclease that have been confirmed biochemically, i.e. canonical bulge–helix–bulge (BHB) RNA substrate (left panel) ( 13 , 27 , 28 ) and non-canonical BHB substrates (right panel). For non-canonical substrates, from the left: a synthetic 4–3–3 and 2–3–3 BHB ( 28 ), a bulge–helix–loop (BHL) ( 29 ) and a trans -spliced BHL formed by two split half tRNA genes ( 13 , 29 ).
    Figure Legend Snippet: Splicing activity and specificity of the NEQ RNA splicing endonuclease. ( A ) A 3′-radiolabeled RNA transcript of the N. equitans tRNA Glu precursor was incubated without enzyme (–) or with either 1 μM NEQ205-NEQ261 splicing endonuclease at 65°C for 20 min alone or followed by incubation with T4 polynucleotide kinase (PNK) and T4 RNA ligase. AFU splicing endonuclease was incubated with a substrate as a positive control. ( B ) Secondary structure of the relaxed BHB motif of N. equitans tRNA Glu precursor. The predicted cleavage sites are indicated by arrows and the CUC anticodon is indicated by a line. ( C ) The mature tRNA product was excised, amplified by RT-PCR and sequenced. The anticodon loop was correctly assembled and the anticodon is underlined. ( D ) Examples of the RNA substrates cleaved by the tRNA splicing endonuclease that have been confirmed biochemically, i.e. canonical bulge–helix–bulge (BHB) RNA substrate (left panel) ( 13 , 27 , 28 ) and non-canonical BHB substrates (right panel). For non-canonical substrates, from the left: a synthetic 4–3–3 and 2–3–3 BHB ( 28 ), a bulge–helix–loop (BHL) ( 29 ) and a trans -spliced BHL formed by two split half tRNA genes ( 13 , 29 ).

    Techniques Used: Activity Assay, Incubation, Positive Control, Amplification, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth"

    Article Title: Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1065

    Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.
    Figure Legend Snippet: Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.

    Techniques Used: Sequencing

    13) Product Images from "Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis"

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    Journal: Scientific Reports

    doi: 10.1038/srep28791

    Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.
    Figure Legend Snippet: Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.

    Techniques Used: Amplification, Isolation, Transgenic Assay, Purification, Polymerase Chain Reaction, Molecular Weight, Marker, Over Expression

    14) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    15) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    16) Product Images from "A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage"

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1265

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .
    Figure Legend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Techniques Used: Ligation

    17) Product Images from "Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth"

    Article Title: Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1065

    Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.
    Figure Legend Snippet: Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.

    Techniques Used: Sequencing

    18) Product Images from "Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh217

    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.
    Figure Legend 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.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Amplification

    19) Product Images from "Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element"

    Article Title: Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

    Journal: RNA

    doi: 10.1261/rna.506607

    Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.
    Figure Legend Snippet: Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.

    Techniques Used: Labeling, Incubation

    The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.
    Figure Legend Snippet: The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.

    Techniques Used: Purification, Labeling, Ligation, Incubation

    20) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea"

    Article Title: Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030229

    Determination of 5′- and 3′-Ends of Haloarchaeal Transcripts (A) Overview of the method. A recently developed method [ 28 ] was used to determine 5′-ends and 3′-ends of haloarchaeal transcripts. The overview schematically shows the different steps of the protocol. Circularization of transcripts with T4 RNA ligase is only possible if their 5′-ends are monophosphorylated, in contrast to the 5′- triphosphate that is present when they are newly synthesized. This led to the initial belief that only processed transcripts can be analyzed [ 28 ], but it turned out that the method is also ideally suited to characterize primary transcripts (compare Discussion ). (B) Sequence of a PCR product representing a transcript with only one specific 3′-end (number 12 in Table 2 ). The stop and start codons of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (C) Sequence of a PCR product representing a transcript with several 3′-ends (number 7 in Table 2 ). Different signal intensities are indicated by lines and the ligation point of the 5′-end and two different 3′-ends are denoted by arrows. (D) Sequences of two clones after cloning the PCR product shown in C (number 7 in Table 2 ). The results of two sequencing reactions of independent clones are shown. In both cases, the stop codon and start codon of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (E) Results after sequencing ten clones. The sequences of five different 3′-ends and their number of occurrence are shown. The 5′-end was found to be identical in all ten cases.
    Figure Legend Snippet: Determination of 5′- and 3′-Ends of Haloarchaeal Transcripts (A) Overview of the method. A recently developed method [ 28 ] was used to determine 5′-ends and 3′-ends of haloarchaeal transcripts. The overview schematically shows the different steps of the protocol. Circularization of transcripts with T4 RNA ligase is only possible if their 5′-ends are monophosphorylated, in contrast to the 5′- triphosphate that is present when they are newly synthesized. This led to the initial belief that only processed transcripts can be analyzed [ 28 ], but it turned out that the method is also ideally suited to characterize primary transcripts (compare Discussion ). (B) Sequence of a PCR product representing a transcript with only one specific 3′-end (number 12 in Table 2 ). The stop and start codons of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (C) Sequence of a PCR product representing a transcript with several 3′-ends (number 7 in Table 2 ). Different signal intensities are indicated by lines and the ligation point of the 5′-end and two different 3′-ends are denoted by arrows. (D) Sequences of two clones after cloning the PCR product shown in C (number 7 in Table 2 ). The results of two sequencing reactions of independent clones are shown. In both cases, the stop codon and start codon of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (E) Results after sequencing ten clones. The sequences of five different 3′-ends and their number of occurrence are shown. The 5′-end was found to be identical in all ten cases.

    Techniques Used: Synthesized, Sequencing, Polymerase Chain Reaction, Ligation, Clone Assay

    22) Product Images from "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)"

    Article Title: Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2009/915061

    Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.
    Figure Legend Snippet: Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.

    Techniques Used: Ligation, Clone Assay, Blocking Assay

    23) Product Images from "Human tRNA-derived small RNAs in the global regulation of RNA silencing"

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing

    Journal: RNA

    doi: 10.1261/rna.2000810

    Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except
    Figure Legend Snippet: Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except

    Techniques Used: Northern Blot, Derivative Assay

    24) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    25) Product Images from "Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay"

    Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

    Journal: RNA

    doi: 10.1261/rna.064501.117

    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).
    Figure Legend Snippet: cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Techniques Used: Labeling, Marker

    26) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    27) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    28) Product Images from "Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro"

    Article Title: Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

    Journal: Molecular and Cellular Biology

    doi:

    T-DNA ligation in vitro is performed by plant enzymes, likely by a DNA ligase. In vitro ligation was performed with nuclear extract from tobacco BY2 cells (A), with extract from pea shoot apices (B), and with the following purified prokaryotic ligases: E. coli DNA ligase (10 U), Taq DNA ligase (40 U), T4 DNA ligase (40 U), and T4 RNA ligase (20 U) (C). (D) Effect of inhibitors (150 μM aphidicolin, 5 μM ddTTP, and 1 mM dTTP) on T-DNA in vitro ligation. Inhibition values represent comparisons of the ligation efficiencies in the presence and absence of the inhibitor. 8-mer–VirD2 (8 pmol) and 8-mer-P (4 pmol) were used as ligation substrates.
    Figure Legend Snippet: T-DNA ligation in vitro is performed by plant enzymes, likely by a DNA ligase. In vitro ligation was performed with nuclear extract from tobacco BY2 cells (A), with extract from pea shoot apices (B), and with the following purified prokaryotic ligases: E. coli DNA ligase (10 U), Taq DNA ligase (40 U), T4 DNA ligase (40 U), and T4 RNA ligase (20 U) (C). (D) Effect of inhibitors (150 μM aphidicolin, 5 μM ddTTP, and 1 mM dTTP) on T-DNA in vitro ligation. Inhibition values represent comparisons of the ligation efficiencies in the presence and absence of the inhibitor. 8-mer–VirD2 (8 pmol) and 8-mer-P (4 pmol) were used as ligation substrates.

    Techniques Used: DNA Ligation, In Vitro, Ligation, Purification, Inhibition

    29) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    30) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    31) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    32) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    33) Product Images from "Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †"

    Article Title: Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1049

    ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.
    Figure Legend Snippet: ( A ) Schematic processing of the p1 16S rRNA. The extra-sequences of 115 nt and 33 nt, flanking the m 16S rRNA at its 5′ and 3′ ends, respectively, are shown on a grey background. RA and FA are the primers used for 3′5′ RACE analysis. The site of annealing of RA to m 16S rRNA, and that of FA to the reverse complement of the m 16S rRNA, are indicated by arrows. Figure not drawn to scale. ( B ) Expected sizes in bp of the RT-PCR products (amplicons) obtained from the different species of 16S rRNA ( p1 , p2 , p3 and m ) by 3′5′ RACE. ( C–F ) Agarose gel electrophoresis of RT-PCR products obtained by 3′5′ RACE from total RNA isolated from MC4100 bacteria grown at 30°C (C), or 44°C (D), or 45°C (E) or 46°C (F). Each RNA sample was thermo-denatured (lanes b), or not (lanes a) prior to the 3′5′ ligation. The sizes (in bp) of the molecular weight markers are indicated to the left of each gel (M). ( G ) The thermodenaturation step dissociates the complementary sequences present at the 3′ and 5′ends of the p1 16S rRNA, and therefore offers to all the 16S rRNA species an equal chance to access to the T4 RNA ligase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Ligation, Molecular Weight

    34) Product Images from "Detection of Head-to-Tail DNA Sequences of Human Bocavirus in Clinical Samples"

    Article Title: Detection of Head-to-Tail DNA Sequences of Human Bocavirus in Clinical Samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019457

    Approach for deciüphering the hairpin like structure of human bocavirus. a: Putative hairpin structures of human bocavirus. In order to test for the self priming capability of the HBoV genome during genome replication different polymerases were added to HBoV DNA isolated from clinical samples. Both, positive strand and negative strand containing isolates were used [23] . Following self-priming elongation the DNA was denaturized and PCR was performed with a single primer. Surprisingly in none of the tested isolates a PCR product was observed, leading to the conclusion that no self priming occurred. b: HBoV end terminus PCR with primer Nrul1 and Ssp1, respectively. HBoV genome preparations were incubated with T7 polymerase and subject to subsequent PCR with primers Boca_end_NruI-1_neu and Boca_end_SspI-1_neu, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in figure 1a . c: HBoV end terminus PCR with primer Nrul2 and Ssp2, respectively. HBoV genome preparations were incubated with Klenow polymerase and subject to subsequent PCR with primers NruI-2 and SspI-2, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in figure 1a . d: HBoV end terminus PCR after circularisation of the genome with primer Nrul2 and Ssp2. HBoV genome preparations were incubated with T4 RNA ligase (self-ligation of single strand genomes and subject to subsequent PCR with primers Nru2 and Ssp2, respectively. Theoretically, head-to-tail sequences should have been amplified provided the terminal regions of the genome allow self-ligation and are not masked by secondary structures resistant to self-ligation. e: HBoV PCR with primer set Boca_end_SspI-1_neu and Boca_end_NruI-1_neu. This PCR approach was performed with primer in the outmost terminal region of the HBoV genome DQ000496. The PCR reaction should have amplified head-to-tail, tail-to-tail, or head-to-head structures, provided the target sequence is present in the clinical isolates. Unfortunately the primers did not bind to a terminal region that is know among all so far published isolates, thus it remains unclear whether primer binding was sufficient.
    Figure Legend Snippet: Approach for deciüphering the hairpin like structure of human bocavirus. a: Putative hairpin structures of human bocavirus. In order to test for the self priming capability of the HBoV genome during genome replication different polymerases were added to HBoV DNA isolated from clinical samples. Both, positive strand and negative strand containing isolates were used [23] . Following self-priming elongation the DNA was denaturized and PCR was performed with a single primer. Surprisingly in none of the tested isolates a PCR product was observed, leading to the conclusion that no self priming occurred. b: HBoV end terminus PCR with primer Nrul1 and Ssp1, respectively. HBoV genome preparations were incubated with T7 polymerase and subject to subsequent PCR with primers Boca_end_NruI-1_neu and Boca_end_SspI-1_neu, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in figure 1a . c: HBoV end terminus PCR with primer Nrul2 and Ssp2, respectively. HBoV genome preparations were incubated with Klenow polymerase and subject to subsequent PCR with primers NruI-2 and SspI-2, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in figure 1a . d: HBoV end terminus PCR after circularisation of the genome with primer Nrul2 and Ssp2. HBoV genome preparations were incubated with T4 RNA ligase (self-ligation of single strand genomes and subject to subsequent PCR with primers Nru2 and Ssp2, respectively. Theoretically, head-to-tail sequences should have been amplified provided the terminal regions of the genome allow self-ligation and are not masked by secondary structures resistant to self-ligation. e: HBoV PCR with primer set Boca_end_SspI-1_neu and Boca_end_NruI-1_neu. This PCR approach was performed with primer in the outmost terminal region of the HBoV genome DQ000496. The PCR reaction should have amplified head-to-tail, tail-to-tail, or head-to-head structures, provided the target sequence is present in the clinical isolates. Unfortunately the primers did not bind to a terminal region that is know among all so far published isolates, thus it remains unclear whether primer binding was sufficient.

    Techniques Used: Isolation, Polymerase Chain Reaction, Incubation, Ligation, Amplification, Sequencing, Binding Assay

    35) Product Images from "Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element"

    Article Title: Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

    Journal: RNA

    doi: 10.1261/rna.506607

    Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.
    Figure Legend Snippet: Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.

    Techniques Used: Labeling, Incubation

    The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.
    Figure Legend Snippet: The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.

    Techniques Used: Purification, Labeling, Ligation, Incubation

    36) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    37) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    38) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    39) Product Images from "A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage"

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq756

    RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.
    Figure Legend Snippet: RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Techniques Used: High Performance Liquid Chromatography, Purification, Expressing, Polyacrylamide Gel Electrophoresis, Ligation, Labeling

    40) Product Images from "Pentatricopeptide Repeat Proteins Stimulate mRNA Adenylation/Uridylation to Activate Mitochondrial Translation in Trypanosomes"

    Article Title: Pentatricopeptide Repeat Proteins Stimulate mRNA Adenylation/Uridylation to Activate Mitochondrial Translation in Trypanosomes

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2011.02.021

    KPAF1 Repression Inhibits Mitochondrial Translation (A) Cloning and sequencing of A/U-tails in CO1 and Cyb mRNAs. Total RNA was circularized with T4 RNA ligase (+), or incubated without ligase (−), and subjected to a single-tube RT-PCR amplification with outward-directed primers specific for 5′ and 3′ mRNA termini. PCR products were separated on 1.4% agarose gel and DNA was extracted from regions indicated by brackets, cloned and sequenced. Representative A/U-tails are shown in 5′-3′ polarity. ), major spots indicated by arrows represent CO1 and Cyb subunits. Cycloheximide-resistant translation produced several additional spots of unknown identity (marked by asterisks) which were also abolished by KPAF1 RNAi.
    Figure Legend Snippet: KPAF1 Repression Inhibits Mitochondrial Translation (A) Cloning and sequencing of A/U-tails in CO1 and Cyb mRNAs. Total RNA was circularized with T4 RNA ligase (+), or incubated without ligase (−), and subjected to a single-tube RT-PCR amplification with outward-directed primers specific for 5′ and 3′ mRNA termini. PCR products were separated on 1.4% agarose gel and DNA was extracted from regions indicated by brackets, cloned and sequenced. Representative A/U-tails are shown in 5′-3′ polarity. ), major spots indicated by arrows represent CO1 and Cyb subunits. Cycloheximide-resistant translation produced several additional spots of unknown identity (marked by asterisks) which were also abolished by KPAF1 RNAi.

    Techniques Used: Clone Assay, Sequencing, Incubation, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Produced

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    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: To this end, DNA ‘anchor’ oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243).

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA
    Article Snippet: T4 RNA ligase is reported to require a single unpaired nucleotide at the 3′-terminus.

    Ligation:

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: .. T4 RNA ligase catalyzes the ligation of an oligonucleotide bearing a 5′ phosphate group (the donor) to a second oligonucleotide bearing a free 3′-OH group (the acceptor). ..

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    Article Snippet: .. The ligation solution contained 5 pg of the synthetic oligonucleotide adapter, 50 mM Tris·HCl (pH 7.8), 10 mM MgCl2 , 1 mM 2-mercaptoethanol, 1 mM ATP, 20 units of human placenta ribonuclease inhibitor (RNasin, Promega), and 20 units of T4 RNA ligase (New England BioLabs). ..

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: .. A typical large-scale ligation reaction using T4 RNA ligase was 40 μM in both RNA fragments in 1× NEB ligation buffer (50 mM Tris–HCl pH = 7.8, 1 mM ATP, 10 mM MgCl2 , 10 mM DTT), 1x in BSA using 5 U T4 RNA ligase per nmol of RNA to be ligated. .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase.

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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 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( 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

    Endoribonucleolytic cleavage with purified H5 protein results in a 3′-OH. A , schematic of potential cleavage products for the TAP-treated (+TAP) or untreated (−TAP) 430-nt ssRNA substrate. I and II , possible scenarios depending on the nature of 3′ ends after cleavage. Ends denoted in boldface type indicate possible results of cleavage. B , schematic of the potential and actual products after treatment of the cleavage reaction with Terminator exonuclease ( Term ). I and II , possible outcomes depending on the nature of 3′ ends after cleavage. Gray line , RNA degraded by Terminator; black line , RNA not degraded by Terminator. C , schematic of the potential and actual products after [5′- 32 P]pCp treatment of unlabeled, purified RNA substrate and products from a cleavage reaction. Black line , RNA labeled with [5′- 32 P]pCp; gray line , RNA not labeled with [5′- 32 P]pCp. Lane 1 , RNA substrate treated with H5 in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 2 , RNA substrate treated with buffer in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 3 , RNA substrate labeled with [5′- 32 P]pCp.

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus *

    doi: 10.1074/jbc.M112.442012

    Figure Lengend Snippet: Endoribonucleolytic cleavage with purified H5 protein results in a 3′-OH. A , schematic of potential cleavage products for the TAP-treated (+TAP) or untreated (−TAP) 430-nt ssRNA substrate. I and II , possible scenarios depending on the nature of 3′ ends after cleavage. Ends denoted in boldface type indicate possible results of cleavage. B , schematic of the potential and actual products after treatment of the cleavage reaction with Terminator exonuclease ( Term ). I and II , possible outcomes depending on the nature of 3′ ends after cleavage. Gray line , RNA degraded by Terminator; black line , RNA not degraded by Terminator. C , schematic of the potential and actual products after [5′- 32 P]pCp treatment of unlabeled, purified RNA substrate and products from a cleavage reaction. Black line , RNA labeled with [5′- 32 P]pCp; gray line , RNA not labeled with [5′- 32 P]pCp. Lane 1 , RNA substrate treated with H5 in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 2 , RNA substrate treated with buffer in cleavage assay, purified, and labeled with [5′- 32 P]pCp; lane 3 , RNA substrate labeled with [5′- 32 P]pCp.

    Article Snippet: This RNA (and RNA not subjected to the cleavage reaction) was then labeled in a reaction containing 150 μCi of [5′-32 P]pCp (3000 Ci/mmol; PerkinElmer Life Sciences) and 10 units of RNA ligase (New England Biolabs) at 4 °C overnight.

    Techniques: Purification, Labeling, Cleavage Assay

    Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.

    Journal: Nucleic Acids Research

    Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide

    doi: 10.1093/nar/gkj474

    Figure Lengend Snippet: Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.

    Article Snippet: Ligation with T4 RNA ligase Synthesized miR173 RNA standard and miR173 with a methyl group on either the 2′ or 3′ OH (miR173-2′OMe or miR173-3′OMe) were treated with calf intestine alkaline phosphatase (New England Biolabs) to remove the 5′P to prevent self-ligation.

    Techniques: Ligation, Activity Assay