t4 rna ligase buffer  (Thermo Fisher)


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    T4 RNA Ligase 10 U µL
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    Thermo Scientific T4 RNA Ligase catalyzes the ATP dependent intra and intermolecular formation of phosphodiester bonds between 5 phosphate and 3 hydroxyl termini of oligonucleotides single stranded RNA and DNA The minimal substrate is a nucleoside 3 5 biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction Applications• RNA 3 end labeling with cytidine 3 5 bis alpha 32P phosphate• Joining RNA to RNA• Synthesis of oligoribonucleotides and oligodeoxyribonucleotides• Specific modifications of tRNAs• Oligodeoxyribonucleotide ligation to single stranded cDNAs for 5 RACE Rapid Amplification of cDNA Ends • Site specific generation of composite primers for PCRNoteThe recommended BSA concentration in the reaction mixture is 0 1mg mL
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    el0021
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    Thermo Fisher t4 rna ligase buffer
    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 <t>T4</t> RNA ligase was omitted. ( C ) The strategy in A ).
    Thermo Scientific T4 RNA Ligase catalyzes the ATP dependent intra and intermolecular formation of phosphodiester bonds between 5 phosphate and 3 hydroxyl termini of oligonucleotides single stranded RNA and DNA The minimal substrate is a nucleoside 3 5 biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction Applications• RNA 3 end labeling with cytidine 3 5 bis alpha 32P phosphate• Joining RNA to RNA• Synthesis of oligoribonucleotides and oligodeoxyribonucleotides• Specific modifications of tRNAs• Oligodeoxyribonucleotide ligation to single stranded cDNAs for 5 RACE Rapid Amplification of cDNA Ends • Site specific generation of composite primers for PCRNoteThe recommended BSA concentration in the reaction mixture is 0 1mg mL
    https://www.bioz.com/result/t4 rna ligase buffer/product/Thermo Fisher
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

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

    Journal: RNA

    doi: 10.1261/rna.5247704

    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 ).
    Figure Legend 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 ).

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Blocking Assay, Ligation

    2) Product Images from "Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges"

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1435248

    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).
    Figure Legend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Techniques Used: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    3) Product Images from "Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges"

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1435248

    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).
    Figure Legend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Techniques Used: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    4) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

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

    Journal: RNA

    doi: 10.1261/rna.5247704

    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 ).
    Figure Legend 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 ).

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Blocking Assay, Ligation

    Related Articles

    Concentration Assay:

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications
    Article Snippet: .. The sample was vortexed and centrifuged before it was treated with T4 RNA ligase (Fermentas; 10 U/µl in storage solution) to give a concentration of 0.25 U/µl (in the final reaction volume). ..

    Incubation:

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges
    Article Snippet: .. Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C. .. To facilitate in vitro circularization, ATP is added to 200 nM, DMSO to 15% (v/v) and T4 RNA ligase 1 to 0.2 U/µl and the reaction is incubated in 250 µl for 16 h at 16°C.

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2
    Article Snippet: .. Precipitated RNA was resuspended in 6.5 µl H2 O and incubated overnight at 16°C after adding 1 µl 10× T4 RNA Ligase Buffer, 1 µl BSA, 1 µl of 20 µM RL5 linker (5′-OH-AGGGAGGACGAUGCGG-OH-3′), and 0.5 µl T4 RNA Ligase. ..

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

    Ligation:

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume. .. To prepare the lariat RNA samples for the assays of Figure 4 , the entire sample was quenched after 15 min with 20 μL of stop solution, and the lariat RNA product was purified by 12% PAGE.

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  • 99
    Thermo Fisher t4 rna ligase buffer
    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by <t>T4</t> RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).
    T4 Rna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/Thermo Fisher
    Average 99 stars, based on 77 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher t4 dna ligase buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Thermo Fisher
    Average 96 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-07
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    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Journal: RNA Biology

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    doi: 10.1080/15476286.2018.1435248

    Figure Lengend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Article Snippet: Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C.

    Techniques: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Synthesis and Labeling of RNA In Vitro

    doi: 10.1002/0471142727.mb0415s102

    Figure Lengend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Article Snippet: 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase.

    Techniques: Radioactivity, Ligation

    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Journal: RNA Biology

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    doi: 10.1080/15476286.2018.1435248

    Figure Lengend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Article Snippet: Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C.

    Techniques: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Article Snippet: For the 3΄-end ligation; adapter ss1 (final conc 10 μM), T4 DNA ligase buffer (40 mM Tris–HCl pH 7.8,10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP), PEG4000 (5% w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free water was added to the sample on ice, in a total volume of 30 μl.

    Techniques: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation