t4 pnk Search Results


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  • 99
    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: 99/100, based on 24281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 polynucleotide kinase
    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by <t>T4</t> polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Enzymatics t4 polynucleotide kinase
    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by <t>T4</t> polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    T4 Polynucleotide Kinase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 94/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 4366 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Roche t4 polynucleotide kinase
    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The <t>T4</t> polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
    T4 Polynucleotide Kinase, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 1330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Bangalore Genei t4 polynucleotide kinase
    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The <t>T4</t> polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
    T4 Polynucleotide Kinase, supplied by Bangalore Genei, used in various techniques. Bioz Stars score: 87/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc t4 polynucleotide kinase
    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The <t>T4</t> polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
    T4 Polynucleotide Kinase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa t4 pnk
    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 <t>T4</t> 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.
    T4 Pnk, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim t4 polynucleotide kinase
    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with <t>T4</t> polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).
    T4 Polynucleotide Kinase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher t4 pnk buffer
    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with <t>T4</t> polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).
    T4 Pnk Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    GE Healthcare t4 polynucleotide kinase
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    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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    86
    TaKaRa 10x t4 pnk buffer
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    10x T4 Pnk Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer t4 polynucleotide kinase
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    T4 Polynucleotide Kinase, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzymatics t4 pnk buffer
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    T4 Pnk Buffer, supplied by Enzymatics, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche t4 pnk buffer
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    T4 Pnk Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 5×t4 pnk buffer a
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
    5×T4 Pnk Buffer A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
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    Roche phosphatase dead t4 pnk
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
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    Sino Biological 10x t4 pnk reaction buffer a
    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by <t>T4</t> polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
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    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and <t>T4</t> PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
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    New England Biolabs t4 pnk dna ligase
    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and <t>T4</t> PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
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    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 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 End Labeled Oligonucleotide Probes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 18427 013 klenow dna polymerase t4 pnk minelute reaction cleanup kit
    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.
    18427 013 Klenow Dna Polymerase T4 Pnk Minelute Reaction Cleanup Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4pnk
    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.
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    Promega t4pnk
    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.
    T4pnk, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific t4 polynucleotide kinase
    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 Polynucleotide Kinase, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate

    Journal: The Journal of Biological Chemistry

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum

    doi: 10.1074/jbc.M110.208306

    Figure Lengend Snippet: Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate

    Article Snippet: After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C.

    Techniques: Polymerase Chain Reaction

    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

    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

    Journal: Nucleic Acids Research

    Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi

    doi: 10.1093/nar/gkr478

    Figure Lengend Snippet: Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

    Article Snippet: Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche).

    Techniques: Clone Assay, Construct, Negative Control, Labeling

    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: T4 PNK consists of four identical subunits of 28.9 kDa each.

    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: T4 PNK consists of four identical subunits of 28.9 kDa each.

    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

    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with T4 polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).

    Journal: Nucleic Acids Research

    Article Title: RecJ exonuclease: substrates, products and interaction with SSB

    doi: 10.1093/nar/gkj503

    Figure Lengend Snippet: RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with T4 polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).

    Article Snippet: Oligonucleotide A was 5′ labeled using T4 polynucleotide kinase and [γ-33 P]ATP and purified by chromatography with G-50 Sephadex (Boehringer Mannheim), phenol/chloroform extraction, ethanol precipitation and resuspended in TE buffer (pH 8.0) or 10 mM Tris–HCl (pH 8.0).

    Techniques: Labeling, Purification

    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

    EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by T4 polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.

    Journal: American journal of physiology. Gastrointestinal and liver physiology

    Article Title: Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1?

    doi: 10.1152/ajpgi.00121.2004

    Figure Lengend Snippet: EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by T4 polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.

    Article Snippet: Briefly, 3.5 pmol of the probe was incubated with 1 µl of T4 polynucleotide kinase 10× buffer (700 mM Tris·HCl, pH 7.6, 100 mM MgCl2 , 50 mM DTT), 1 µl of [γ-32 P]ATP (3,000 Ci/mmol at 10 mCi/ml, Amersham Biosciences, Piscataway, NJ), and 1 µl of T4 polynucleotide kinase (5–10 U/µl) in a 10 µl reaction at 37°C for 10 min.

    Techniques: Binding Assay, Labeling, Electrophoretic Mobility Shift Assay, Incubation

    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

    doi: 10.1016/j.omtn.2017.07.008

    Figure Lengend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Article Snippet: The protocol was modified as follows when CIP or T4 PNK treatment is necessary: (1) for CIP treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of CIP, 4 μL of 10× CutSmart buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min; and (2) for T4 PNK treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of T4 PNK, 4 μL of 10× T4 PNK buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min. All T7 in vitro transcription products were purified by Micro Bio-Spin P-30 Gel Columns, Tris Buffer, from Bio-Rad.

    Techniques: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

    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