t4 rna ligase Thermo Fisher Search Results


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  • 99
    Thermo Fisher ambion t4 rna ligase
    <t>RNA</t> preparation and purification. MicroRNA precursors were synthesized using T7 polymerase. (A) Following transcription, each RNA was resolved by UREA-PAGE ( eg , pre-let-7a-2), gel-extracted and tested again for integrity by denaturing PAGE (left and right). (B) While the RNA molecules aligned, two major folding types appeared: a precursor RNA dimer (B), and a precursor RNA hairpin (C). (D) Following denaturing gel extraction, we resolved all <t>RNAs</t> by Native-PAGE and only stem-loop precursor structures were gel-extracted (left). Each hairpin structure was tested again by Native-PAGE to confirm that did not form precursor RNA dimers (right; pre-let-7a-2 and pre-let-7b are shown as examples). To test whether dimer structures could be avoided, samples were heated to 95 °C and slowly cooled down to RT (B, boiled; N, not boiled). Heating the samples did not disrupt RNA dimer structures but, in contrast, promoted their formation.
    Ambion T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 rna ligase
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher rna adaptor
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    Rna Adaptor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher rna tags
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    Rna Tags, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher 10x t4 rna ligase buffer
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    10x T4 Rna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 rna ligase buffer 10×
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase Buffer 10×, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pierce rna 3 end biotinylation kit
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    Pierce Rna 3 End Biotinylation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 dna ligase rna ligase 2
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Dna Ligase Rna Ligase 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 rna ligase 2 truncated
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase 2 Truncated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 dna ligase
    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using <t>T4</t> DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 dna ligase buffer 5×
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Ligase Buffer 5×, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 dna ligase 5 u µl
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Ligase 5 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mirna 3 0 genechip arrays
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Mirna 3 0 Genechip Arrays, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads m 280 streptavidin
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Dynabeads M 280 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RNA preparation and purification. MicroRNA precursors were synthesized using T7 polymerase. (A) Following transcription, each RNA was resolved by UREA-PAGE ( eg , pre-let-7a-2), gel-extracted and tested again for integrity by denaturing PAGE (left and right). (B) While the RNA molecules aligned, two major folding types appeared: a precursor RNA dimer (B), and a precursor RNA hairpin (C). (D) Following denaturing gel extraction, we resolved all RNAs by Native-PAGE and only stem-loop precursor structures were gel-extracted (left). Each hairpin structure was tested again by Native-PAGE to confirm that did not form precursor RNA dimers (right; pre-let-7a-2 and pre-let-7b are shown as examples). To test whether dimer structures could be avoided, samples were heated to 95 °C and slowly cooled down to RT (B, boiled; N, not boiled). Heating the samples did not disrupt RNA dimer structures but, in contrast, promoted their formation.

    Journal: Acta Pharmacologica Sinica

    Article Title: First step in pre-miRNAs processing by human Dicer

    doi: 10.1038/aps.2009.108

    Figure Lengend Snippet: RNA preparation and purification. MicroRNA precursors were synthesized using T7 polymerase. (A) Following transcription, each RNA was resolved by UREA-PAGE ( eg , pre-let-7a-2), gel-extracted and tested again for integrity by denaturing PAGE (left and right). (B) While the RNA molecules aligned, two major folding types appeared: a precursor RNA dimer (B), and a precursor RNA hairpin (C). (D) Following denaturing gel extraction, we resolved all RNAs by Native-PAGE and only stem-loop precursor structures were gel-extracted (left). Each hairpin structure was tested again by Native-PAGE to confirm that did not form precursor RNA dimers (right; pre-let-7a-2 and pre-let-7b are shown as examples). To test whether dimer structures could be avoided, samples were heated to 95 °C and slowly cooled down to RT (B, boiled; N, not boiled). Heating the samples did not disrupt RNA dimer structures but, in contrast, promoted their formation.

    Article Snippet: For 3′ end labeled RNAs, RNA ligase (Ambion, Austin, TX) and [α-32 P] pCp were used according to the manufacturer's recommendations.

    Techniques: Purification, Synthesized, Polyacrylamide Gel Electrophoresis, Gel Extraction, Clear Native PAGE

    5′-RACE analysis of the amiR-guided mRNA cleavage

    Journal: Plant biology (Stuttgart, Germany)

    Article Title: Efficient transformation and artificial miRNA gene silencing in Lemna minor

    doi: 10.1111/plb.12215

    Figure Lengend Snippet: 5′-RACE analysis of the amiR-guided mRNA cleavage

    Article Snippet: 2–5 μg of total RNA were used for the 5′RACE adapter ligation reaction (T4 RNA ligase 5 U/μl from Ambion).

    Techniques:

    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Journal: RNA Biology

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    doi: 10.1080/15476286.2018.1518854

    Figure Lengend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Article Snippet: RNA was resuspended in 14.5 µl H2 O and subjected to T4 RNA Ligase reaction by adding 1 µl of 20 µM 5′-phosporylated RL3 (5′-P -GUGUCAGUCACUUCCAGCGG-Puromycin-3′), 2 µl 10× T4 Ligase Buffer, 2 µl BSA, 0.5 µl T4 RNA Ligase (ThermoFisher), and incubated overnight at 16°C.

    Techniques: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: The solution was brought to 100 μL total volume containing 1× T4 RNA ligase buffer (see above), 50 μM ATP, and 40–200 units of T4 RNA ligase (MBI Fermentas).

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Article Snippet: The solution was brought to 100 μL total volume containing 1× T4 RNA ligase buffer (see above), 50 μM ATP, and 40–200 units of T4 RNA ligase (MBI Fermentas).

    Techniques: In Vitro

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Article Snippet: The solution was brought to 100 μL total volume containing 1× T4 RNA ligase buffer (see above), 50 μM ATP, and 40–200 units of T4 RNA ligase (MBI Fermentas).

    Techniques: Blocking Assay, Ligation

    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Journal: Chembiochem

    Article Title: Practical Synthesis of Cap‐4 RNA

    doi: 10.1002/cbic.201900590

    Figure Lengend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Article Snippet: The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. .

    Techniques: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    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: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

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

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. 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. 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. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    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 treated with CIAP. 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. 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. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

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

    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: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    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: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

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

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    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: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

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

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. 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. 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. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    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 treated with CIAP. 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. 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. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

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

    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: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    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: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

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