t4 polynucleotide kinase Search Results


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  • 95
    New England Biolabs t4 pnk
    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or <t>T4</t> PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore t4 pnk
    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
    T4 Pnk, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    New England Biolabs t4 pnk buffer
    Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and <t>T4</t> PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.
    T4 Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 polynucleotide kinase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and <t>T4</t> polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7876 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t4 polynucleotide kinase
    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using <t>T4</t> polynucleotide kinase. RNAs in
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 24211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Promega t4 polynucleotide kinase
    Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using <t>T4</t> polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.
    T4 Polynucleotide Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 4366 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Roche t4 polynucleotide kinase
    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 <t>T4</t> polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).
    T4 Polynucleotide Kinase, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 1332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    TaKaRa t4 polynucleotide kinase
    Preparation and in vitro activity of dumbbell-shape RNA with endless nucleotide. RNA was prepared as described under method in supplementary materials and the RNA 5′ end was phosphorylated by treating with <t>T4</t> polynucleotide and the RNA 3′ and 5′ ends were ligated and converted to a circular type by treating with T4 RNA ligase (A). The product was confirmed by gel electrophoresis (B). HCT 116 cell lines were treated with varying concentrations of each RNA in the presence of lipofectamine and after 48 h, mRNA was extracted and the amount of hGAPDH was quantified by real time PCR (C). Data are expressed as the mean ± s.e.m. *p
    T4 Polynucleotide Kinase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 2095 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Toyobo t4 polynucleotide kinase
    ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and <t>T4</t> polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).
    T4 Polynucleotide Kinase, supplied by Toyobo, used in various techniques. Bioz Stars score: 96/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim t4 polynucleotide kinase
    ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and <t>T4</t> polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).
    T4 Polynucleotide Kinase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Epicentre Biotechnologies t4 polynucleotide kinase
    ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and <t>T4</t> polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).
    T4 Polynucleotide Kinase, supplied by Epicentre Biotechnologies, used in various techniques. Bioz Stars score: 96/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
    T4 Polynucleotide Kinase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 1832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Euromedex t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    92
    SibEnzyme t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Stratagene t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    96
    PerkinElmer t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Enzymatics t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    92
    Fisher Scientific t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Bangalore Genei t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Transgenomic t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Illumina Inc t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Lofstrand t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Genecraft t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Cell Signaling Technology Inc t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Enzymax t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    AB ANALITICA Srl t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Kaneka Corp t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    Epicentre Technologies Corp t4 polynucleotide kinase
    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using <t>T4</t> polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands
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    N/A
    T4 Polynucleotide Kinase T4 PNK catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5 hydroxyl terminus of polynucleotides double and single stranded DNA
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    Image Search Results


    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Journal: Nature Communications

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    doi: 10.1038/s41467-017-00484-w

    Figure Lengend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Article Snippet: Samples were treated with CIP or T4 PNK by addition of “Cutsmart” or “PNK” buffer from New England Biolabs and 10 units of CIP or T4 PNK and incubation at 37 °C for 15 min. Mock treated samples contained only Cutsmart buffer and water in lieu of CIP or T4 PNK.

    Techniques: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Journal: Nature Communications

    Article Title: Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    doi: 10.1038/ncomms9430

    Figure Lengend Snippet: Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Article Snippet: To measure the half-life of saiRNA with terminal 2′, 3′-cyclic phosphate or hydroxyl groups, T7-transcribed saiRNA-RZs treated or untreated with T4 PNK were incubated with Ago2-KO 293 cell lysates in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 1% NP-40, 0.5% sodium deoxycholate (Sigma), 0.1 U μl−1 RNase Inhibitor and 1/100 protease inhibitor cocktail) for different time periods.

    Techniques: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, shRNA, Luciferase, Northern Blot, Western Blot, Transfection, Activity Assay, Stable Transfection, Transduction

    Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Journal: JCI Insight

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    doi: 10.1172/jci.insight.127317

    Figure Lengend Snippet: Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Article Snippet: To half of the eluted exRNA, i.e., 14 μl, we added 6 μl of a master mix corresponding to the equivalent of 2 μl ×10 T4 PNK buffer, 2 μl 10 mM ATP, 1 μl RNase-free water, and 1 μl T4 PNK (NEB, catalog M0201S) for a final reaction volume of 20 μl in a 1.5 ml siliconized microcentrifuge tube.

    Techniques:

    Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads  > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from  n  = 6 individual samples per condition.

    Journal: JCI Insight

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    doi: 10.1172/jci.insight.127317

    Figure Lengend Snippet: Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from n = 6 individual samples per condition.

    Article Snippet: To half of the eluted exRNA, i.e., 14 μl, we added 6 μl of a master mix corresponding to the equivalent of 2 μl ×10 T4 PNK buffer, 2 μl 10 mM ATP, 1 μl RNase-free water, and 1 μl T4 PNK (NEB, catalog M0201S) for a final reaction volume of 20 μl in a 1.5 ml siliconized microcentrifuge tube.

    Techniques: RNA Sequencing Assay, Isolation, Purification

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkq572

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

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

    Techniques: Ligation, Purification, Sequencing, Blocking Assay

    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Journal: Genome Research

    Article Title: Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

    doi: 10.1101/gr.121426.111

    Figure Lengend Snippet: Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Article Snippet: Total RNA was isolated using TRIzol (Invitrogen), and small RNA samples were 5′ labeled by first treating with calf alkaline phosphatase (Roche) followed by phosphorylation with T4 polynucleotide kinase (NEB) and 32 P-γ-ATP.

    Techniques: Labeling, Molecular Weight

    Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

    Journal: Journal of Bacteriology

    Article Title: Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †

    doi: 10.1128/JB.00011-07

    Figure Lengend Snippet: Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

    Article Snippet: The purified DNA fragments (3.5 pmol) were end labeled with 10 U of T4 polynucleotide kinase (Promega) and 10 μCi of [γ32 -P]ATP (Perkin-Elmer, Wellesley, MA) at 37°C for 10 min.

    Techniques: In Vivo, Binding Assay, Sequencing, Labeling, End Labeling, Polyacrylamide Gel Electrophoresis

    Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate. A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ 32P ] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4 ). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ 32P ] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18] . The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.

    Journal: PLoS Genetics

    Article Title: Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

    doi: 10.1371/journal.pgen.1001225

    Figure Lengend Snippet: Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate. A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ 32P ] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4 ). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ 32P ] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18] . The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.

    Article Snippet: For detection of pre-rRNA precursors and small RNA, 10 pmoles of oligonucleotide primers were 5′end labeled using 50 µCi of [γ32 P] ATP (6000 Ci/mmol) and T4 PNK (Promega).

    Techniques: Mutagenesis, Northern Blot, Isolation, Labeling

    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).

    Journal: RNA

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

    doi: 10.1261/rna.064501.117

    Figure Lengend 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).

    Article Snippet: For 5′-end labeling, degraded RNA fragments, γ-P32 -ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32 ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min.

    Techniques: Labeling, Marker

    Preparation and in vitro activity of dumbbell-shape RNA with endless nucleotide. RNA was prepared as described under method in supplementary materials and the RNA 5′ end was phosphorylated by treating with T4 polynucleotide and the RNA 3′ and 5′ ends were ligated and converted to a circular type by treating with T4 RNA ligase (A). The product was confirmed by gel electrophoresis (B). HCT 116 cell lines were treated with varying concentrations of each RNA in the presence of lipofectamine and after 48 h, mRNA was extracted and the amount of hGAPDH was quantified by real time PCR (C). Data are expressed as the mean ± s.e.m. *p

    Journal: PLoS ONE

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents

    doi: 10.1371/journal.pone.0042655

    Figure Lengend Snippet: Preparation and in vitro activity of dumbbell-shape RNA with endless nucleotide. RNA was prepared as described under method in supplementary materials and the RNA 5′ end was phosphorylated by treating with T4 polynucleotide and the RNA 3′ and 5′ ends were ligated and converted to a circular type by treating with T4 RNA ligase (A). The product was confirmed by gel electrophoresis (B). HCT 116 cell lines were treated with varying concentrations of each RNA in the presence of lipofectamine and after 48 h, mRNA was extracted and the amount of hGAPDH was quantified by real time PCR (C). Data are expressed as the mean ± s.e.m. *p

    Article Snippet: The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA.

    Techniques: In Vitro, Activity Assay, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Article Snippet: The positive control was oligo 11 phosphorylated by T4 PNK.

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Article Snippet: The positive control was oligo 11 phosphorylated by T4 PNK.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and T4 polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).

    Journal: Nucleic Acids Research

    Article Title: Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm

    doi: 10.1093/nar/gki309

    Figure Lengend Snippet: ( A ) Two-dimensional thin layer chromatography of modified nucleosides at position 9 of mitochondrial tRNAs. Open circles indicate spots of nucleotides (A, U, G and C bearing 5′-phosphate) detected by UV shadowing. Open and filled triangles indicate m 1 A and m 6 A [which is considered to be converted form of m 1 A ( 21 )], respectively. Nucleotide analysis was performed as follows. Each 3′ fragment generated by alkaline digestion of the tRNA was labeled at its 5′ terminus with [γ- 32 P]ATP and T4 polynucleotide kinase. Labeled RNAs were loaded onto 10% denaturing polyacrylamide gel and each 5′-labeled 3′-fragment was excised from the gel. The fragments were then digested with P 1 nuclease and the resulting 5′-labeled mononucleotides were analyzed by TLC. 2D-TLC analyses were performed using the solvents; isobutyric acid/concentrated ammonia/water (66:1:33, v/v/v) for the first dimension in both systems, 2-propanol/HCl/water (70:15:15, v/v/v) for the second dimension in system A, and ammonium sulfate/0.1 M sodium phosphate, pH 6.8/1-propanol (60 g:100 ml:2 ml) for the second dimension in system B. 5′-nucleotides of tRNAs were also detected by TLC because each 5′-fragment generated by alkaline digestion of the tRNA was also labeled at its 5′-terminus by the phosphorylation reaction described above (although the 5′-fragment had 5′-phosphate even before the reaction, the 5′-phosphate may have exchanged with labeled phosphate of [γ- 32 P]ATP), and the 5′-labeled 5′-fragments migrated together with the 5′-labeled 3′-fragments on the gel. ( B ) Nucleotide sequences of A.suum mt tRNAs. Abbreviations are the same as in Table 1 . Asterisks (*) show modified uridines, whose details will be described in another manuscript (Sakurai et al ., in preparation).

    Article Snippet: Preparation of RNA fragments, 5′ end phosphorylation using T4 polynucleotide kinase (Toyobo), 3′ end-nucleoside deprivation using NaIO4 , and dephosphorylation using E.coli alkaline phosphatase (BAP) (Takara Shuzo) were performed as described ( ).

    Techniques: Thin Layer Chromatography, Modification, Generated, Labeling

    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using T4 polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands

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

    Article Title: Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase

    doi: 10.1073/pnas.0730835100

    Figure Lengend Snippet: RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using T4 polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands

    Article Snippet: Ultrapure dNTP, 2′,3′-dideoxynucleoside triphosphate, and T4 polynucleotide kinase were purchased from Amersham Pharmacia; RNaseA, CR, deoxycytidine (CdR), uridine, and deoxyuridine were from Sigma; T7 sequenase (version 2.0) and RNase inhibitor were from United States Biochemical; and uracil DNA glycosylase (UDG) and apurinic endonuclease (APE) were generous gifts from D. Mosbaugh (Oregon State University, Corvallis).

    Techniques: Western Blot, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

    Scheme depicting the overall strategy for the construction of smart mutant library by molecular shuffling of active mutants. ( A ) Mutagenic oligonucleotides containing the codon for PCR active single mutants were pooled together and phosphorylated at 5′ end with T4 polynucleotide kinase. ( B , C ) A single strand transient DNA template of the target gene (KlenTaq DNA polymerase) was prepared by PCR in the presence of dUTP and subsequent digestion by λ exonuclease. ( D , E ) The mutagenic oligonucleotides were annealed and the gaps and nicks in the chimeric strand were filled and ligated. ( F ) The transient DNA template was cleaved and the remaining chimeric sequences were PCR amplified. ( G ) The recombinant plasmids were transformed into  E. coli  and grown on selection plate for the generation of combinatorial library.

    Journal: Scientific Reports

    Article Title: Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

    doi: 10.1038/s41598-018-37233-y

    Figure Lengend Snippet: Scheme depicting the overall strategy for the construction of smart mutant library by molecular shuffling of active mutants. ( A ) Mutagenic oligonucleotides containing the codon for PCR active single mutants were pooled together and phosphorylated at 5′ end with T4 polynucleotide kinase. ( B , C ) A single strand transient DNA template of the target gene (KlenTaq DNA polymerase) was prepared by PCR in the presence of dUTP and subsequent digestion by λ exonuclease. ( D , E ) The mutagenic oligonucleotides were annealed and the gaps and nicks in the chimeric strand were filled and ligated. ( F ) The transient DNA template was cleaved and the remaining chimeric sequences were PCR amplified. ( G ) The recombinant plasmids were transformed into E. coli and grown on selection plate for the generation of combinatorial library.

    Article Snippet: Reactions (50 μl) containing 1X T4 PNK buffer, 0.4 μM of oligonucleotides, 0.4 U/μl T4 PNK and 0.4 μC/μl [γ-32 P]-ATP were incubated at 37 °C for 60 min and stopped by incubating at 95 °C for 2 min. After removal of excess ATP and additional salts through gel filtration by Sephadex G-25 microspin column (GE Healthcare), 20 μl of unlabelled primer (10 μM) was added to get a final concentration of 3 μM diluted radioactive labelled primer for use in primer extension experiments.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Recombinant, Transformation Assay, Selection