t4 rna ligase buffer  (Thermo Fisher)


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    Name:
    T4 RNA Ligase 10 U µL
    Description:
    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
    Catalog Number:
    el0021
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    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 90 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-02
    90/100 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

    Clone Assay:

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Briefly, 3 to 5 μg of total RNA was ligated to 300 ng of 5′ RACE adaptor using 5 units of T4 RNA ligase (Ambion, Austin, TX) for 1 h at 37°C. .. The resulting PCR products were cloned into pCR2.1-TOPO and sequenced using the M13rev primer.

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
    Article Snippet: Paragraph title: Small RNA Isolation and Cloning ... Briefly, small RNAs were polyadenylated, and a 5′-adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase (Invitrogen).

    Amplification:

    Article Title: Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus
    Article Snippet: Then they were ligated to 500 pmol 5′ RNA adapter (5′-CAGACUGGAUCCGUCGUC-3′; Integrated DNA Technologies) at their 5′ ends by 50 units of T4 RNA ligase (Ambion) at 17°C for 12 h. Adaptor-ligated RNA was precipitated again with isopropanol, and 2 μg served as the template for reverse-transcription (RT). .. Then a 2 μL aliquot of the RT reaction was used for the first-run PCR amplification with 20 pmol of each RT-primer and adapter-specific primer; and 1 μL aliquot of products served as the template of the second round PCR using 20 pmol adapter-specific and a gene-specific nested primer (R primer in ) upstream of the RT-primer.

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    Article Snippet: The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). .. Templates for in vitro transcription of HIV-2 RNA molecules were obtained by PCR amplification of fragments from the HIV-2 plasmid pROD10-EVA232 using a forward primer containing a T7 promoter sequence (Additional file ).

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
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    Synthesized:

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    Article Snippet: The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). .. RNAs were synthesized using T7-MEGAscript (Ambion) and purified using Direct-zol™ RNA MiniPrep (Zymo Research).

    Neutralization:

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    Article Snippet: Neutralization and small fragment removal was performed with water-saturated G50 columns (Ambion NucAway). .. The recovered single stranded cDNA was dried to near completion using a Servo SpeedVac, followed by a second 5′-adenylated linker addition (IDTDNA – Linker 2) to the cDNA using T4 RNA ligase (Ambion).

    TA Cloning:

    Article Title: Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
    Article Snippet: In order to sequence the 3′ and 5′ ends of the BDV genome, a purified synthetic RNA transcript of known sequence was ligated to the 3′ and 5′ ends of the BDV genomic RNA with T4 RNA ligase (Life Technologies). .. These PCR products were purified and ligated into the pCR 2.1 vector (TA cloning kit; Invitrogen) and sequenced with the Dye Primer cycle-sequencing kit (Applied Biosystems) by using the M13 Reverse and M13 Forward (−40) primer.

    Quantitative RT-PCR:

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Paragraph title: Real-time RT-PCR and 5′ RACE. ... Briefly, 3 to 5 μg of total RNA was ligated to 300 ng of 5′ RACE adaptor using 5 units of T4 RNA ligase (Ambion, Austin, TX) for 1 h at 37°C.

    SYBR Green Assay:

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: All PCRs were performed on either an ABI Prism 7000 real-time PCR detection system (Applied Biosystems, Carlsbad, CA) or an Eppendorf Mastercycler ep realplex 2 apparatus (Eppendorf, Hauppauge, NY) with PerfeCTa SYBR green FastMix, ROX (Quanta Biosciences, Gaithersburg, MD), and gene-specific primers. .. Briefly, 3 to 5 μg of total RNA was ligated to 300 ng of 5′ RACE adaptor using 5 units of T4 RNA ligase (Ambion, Austin, TX) for 1 h at 37°C.

    cDNA Library Assay:

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    Incubation:

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    Article Snippet: Following RT, Exonuclease I (EXO1, Thermo) was added to the RT reaction mixture, and incubated for 30 min to remove excess primer. .. The recovered single stranded cDNA was dried to near completion using a Servo SpeedVac, followed by a second 5′-adenylated linker addition (IDTDNA – Linker 2) to the cDNA using T4 RNA ligase (Ambion).

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: .. For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight. .. Unincorporated 32 P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare).

    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. ..

    Activity Assay:

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: .. In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo). ..

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2
    Article Snippet: To generate a substrate for single-strand RNA-specific activity, poly(C) (Sigma P4903) was resuspended in 40 mM Tris-acetate pH 8.3, 100 mM potassium acetate, 30 mM magnesium acetate, and incubated 10 min at 95°C to fragment the polymer. .. 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.

    Western Blot:

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: The purity of the enzymes was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and by western blot using anti-GST antibodies, revealing > 90% homogeneity. .. In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo).

    Over Expression:

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: Paragraph title: Overexpression, purification and in vitro activity assays of human Dis3 isoforms ... In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo).

    Hybridization:

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: .. For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight. .. Unincorporated 32 P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare).

    Chromatography:

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight. .. Unincorporated 32 P was removed by exclusion chromatography (Illustra Microspin G-25 column, GE Healthcare).

    Ligation:

    Article Title: Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae
    Article Snippet: .. 5' RNA linker ligation RNA pellets were resuspended in 10 µl of ligation mix (50 mM Tris pH 7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml bovine serum albumin, 2 µM 5' RNA linker, 1 U/µl T4 RNA ligase (Fermentas EL0021), 1 U/µl SUPERase·In, 1 0% DMSO) and incubated at 15°C for 2 h. .. RNA size selection Ligation reaction was terminated by adding 10 µl of 2× formamide gel loading buffer (Invitrogen AM8546G), heated for 2 minutes at 70°C and then quickly chilled on ice.

    Article Title: Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves
    Article Snippet: .. The cleaved products were uncapped and carried a free phosphate, thereby allowing direct ligation with the RNA adaptor RA44 using T4 RNA Ligase (Ambion, USA). .. The ligation products were extracted using phenol/chloroform and precipitated with glycogen before first-strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Invitrogen, USA).

    Article Title: Characterization of phosphorus-regulated miR399 and miR827 and their isomirs in barley under phosphorus-sufficient and phosphorus-deficient conditions
    Article Snippet: .. Briefly, poly(A) RNA was extracted from total RNA of barley plants using the Oligotex kit (Qiagen) and then ligated with a 5′ RNA adaptor containing a Mme I restriction site using T4 RNA ligase (Invitrogen), followed by reverse transcription, second-strand synthesis, Mm eI digestion, ligation of a 3′ dsDNA adaptor, gel-purification, and PCR amplification. .. Amplified PCR products were sequenced with the Illumina HiSeq platform.

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
    Article Snippet: Briefly, small RNAs were polyadenylated, and a 5′-adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase (Invitrogen). .. The ligation products were reverse transcribed to produce small RNA cDNAs, which were then amplified using PCR.

    Inhibition:

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: For RNA complex inhibition assay with ORNs, the simultaneous transcription of S7-S10 (combination of 3 or 4) was performed in the presence or absence of 20 pmol of S10.1, S10.2, S10.4, S10.5 and Scr ORNs and analysed as described above. .. For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight.

    Polymerase Chain Reaction:

    Article Title: Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus
    Article Snippet: Then they were ligated to 500 pmol 5′ RNA adapter (5′-CAGACUGGAUCCGUCGUC-3′; Integrated DNA Technologies) at their 5′ ends by 50 units of T4 RNA ligase (Ambion) at 17°C for 12 h. Adaptor-ligated RNA was precipitated again with isopropanol, and 2 μg served as the template for reverse-transcription (RT). .. Then a 2 μL aliquot of the RT reaction was used for the first-run PCR amplification with 20 pmol of each RT-primer and adapter-specific primer; and 1 μL aliquot of products served as the template of the second round PCR using 20 pmol adapter-specific and a gene-specific nested primer (R primer in ) upstream of the RT-primer.

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: Reaction conditions consisted of 40 rounds of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s. Single PCR products were confirmed by performing a postamplification melt curve analysis for each reaction. .. Briefly, 3 to 5 μg of total RNA was ligated to 300 ng of 5′ RACE adaptor using 5 units of T4 RNA ligase (Ambion, Austin, TX) for 1 h at 37°C.

    Article Title: Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing
    Article Snippet: 5' RNA adaptor (5'-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3') was ligated to the purified mRNA by T4 RNA ligase (Ambion), followed by a reverse transcription reaction. .. The reverse transcription product was amplified using 5' RNA adaptor primer (5'-GCACGAGGACACTGACATGGACTGA-3') and gene specific primers for 30 cycles of PCR.

    Article Title: Characterization of phosphorus-regulated miR399 and miR827 and their isomirs in barley under phosphorus-sufficient and phosphorus-deficient conditions
    Article Snippet: .. Briefly, poly(A) RNA was extracted from total RNA of barley plants using the Oligotex kit (Qiagen) and then ligated with a 5′ RNA adaptor containing a Mme I restriction site using T4 RNA ligase (Invitrogen), followed by reverse transcription, second-strand synthesis, Mm eI digestion, ligation of a 3′ dsDNA adaptor, gel-purification, and PCR amplification. .. Amplified PCR products were sequenced with the Illumina HiSeq platform.

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: HIV-1 TAR RNA and unmodified human tRNALys3 (referred to here as tRNALys3 ) were obtained using a PCR-generated template (Additional file ) and Ambion T7-MEGAshortscript. .. The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare).

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
    Article Snippet: Briefly, small RNAs were polyadenylated, and a 5′-adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase (Invitrogen). .. The ligation products were reverse transcribed to produce small RNA cDNAs, which were then amplified using PCR.

    Article Title: Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
    Article Snippet: In order to sequence the 3′ and 5′ ends of the BDV genome, a purified synthetic RNA transcript of known sequence was ligated to the 3′ and 5′ ends of the BDV genomic RNA with T4 RNA ligase (Life Technologies). .. The regions around the 5′ and 3′ ends were amplified by PCR with specific primers that anneal to the ligated synthetic RNA and the BDV genome sequences.

    Gel Purification:

    Article Title: Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species
    Article Snippet: A second gel purification was performed as above, excising the gel fragment above the XC dye band to remove excess 3′ linker. .. The recovered single stranded cDNA was dried to near completion using a Servo SpeedVac, followed by a second 5′-adenylated linker addition (IDTDNA – Linker 2) to the cDNA using T4 RNA ligase (Ambion).

    Article Title: Characterization of phosphorus-regulated miR399 and miR827 and their isomirs in barley under phosphorus-sufficient and phosphorus-deficient conditions
    Article Snippet: .. Briefly, poly(A) RNA was extracted from total RNA of barley plants using the Oligotex kit (Qiagen) and then ligated with a 5′ RNA adaptor containing a Mme I restriction site using T4 RNA ligase (Invitrogen), followed by reverse transcription, second-strand synthesis, Mm eI digestion, ligation of a 3′ dsDNA adaptor, gel-purification, and PCR amplification. .. Amplified PCR products were sequenced with the Illumina HiSeq platform.

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2
    Article Snippet: The reaction was resolved on a denaturing polyacrylamide gel and poly(C) chains of ~100 nucleotides were isolated by gel-purification and radiolabeled by PNK-treatment. .. 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.

    Nucleic Acid Electrophoresis:

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: Transcripts were purified by denaturing gel electrophoresis (8 M urea) in 1 × TBE, followed by elution and ethanol precipitation. .. The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare).

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: The purity of the enzymes was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and by western blot using anti-GST antibodies, revealing > 90% homogeneity. .. In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo).

    Combined Bisulfite Restriction Analysis Assay:

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2
    Article Snippet: Paragraph title: Purification of RNase V1 from cobra venom ... 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.

    Isolation:

    Article Title: Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves
    Article Snippet: In brief, poly (A) + RNA was isolated from cucumber leaves using a magnetic mRNA isolation kit (NEB, UK). .. The cleaved products were uncapped and carried a free phosphate, thereby allowing direct ligation with the RNA adaptor RA44 using T4 RNA Ligase (Ambion, USA).

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
    Article Snippet: Paragraph title: Small RNA Isolation and Cloning ... Briefly, small RNAs were polyadenylated, and a 5′-adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase (Invitrogen).

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2
    Article Snippet: This partially complementary/doubled-stranded substrate (1 nmol) was digested with indicated RNase V1 fractions in a 20 µl-reaction as stated above, and RNA was isolated by phenol-chloroform extraction. .. 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.

    Electrophoretic Mobility Shift Assay:

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: Paragraph title: In vitro transcription for RNA-RNA interaction assays, RNA-RNA interaction in the presence of ORN and electrophoretic mobility shift assay ... For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight.

    Purification:

    Article Title: Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing
    Article Snippet: .. 5' RNA adaptor (5'-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3') was ligated to the purified mRNA by T4 RNA ligase (Ambion), followed by a reverse transcription reaction. .. The reverse transcription product was amplified using 5' RNA adaptor primer (5'-GCACGAGGACACTGACATGGACTGA-3') and gene specific primers for 30 cycles of PCR.

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: .. The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). .. Templates for in vitro transcription of HIV-2 RNA molecules were obtained by PCR amplification of fragments from the HIV-2 plasmid pROD10-EVA232 using a forward primer containing a T7 promoter sequence (Additional file ).

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: Paragraph title: Overexpression, purification and in vitro activity assays of human Dis3 isoforms ... In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo).

    Article Title: Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
    Article Snippet: .. In order to sequence the 3′ and 5′ ends of the BDV genome, a purified synthetic RNA transcript of known sequence was ligated to the 3′ and 5′ ends of the BDV genomic RNA with T4 RNA ligase (Life Technologies). .. The RNA was reverse transcribed by using random hexamer primers.

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2
    Article Snippet: Paragraph title: Purification of RNase V1 from cobra venom ... 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.

    Sequencing:

    Article Title: Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves
    Article Snippet: The cleaved products were uncapped and carried a free phosphate, thereby allowing direct ligation with the RNA adaptor RA44 using T4 RNA Ligase (Ambion, USA). .. The amplicons were further confirmed by sequencing.

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). .. Templates for in vitro transcription of HIV-2 RNA molecules were obtained by PCR amplification of fragments from the HIV-2 plasmid pROD10-EVA232 using a forward primer containing a T7 promoter sequence (Additional file ).

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
    Article Snippet: Briefly, small RNAs were polyadenylated, and a 5′-adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase (Invitrogen). .. The PCR products were directly subcloned into pcDNA3.1 TOPO vector (Invitrogen) for sequencing analysis.

    Article Title: Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
    Article Snippet: .. In order to sequence the 3′ and 5′ ends of the BDV genome, a purified synthetic RNA transcript of known sequence was ligated to the 3′ and 5′ ends of the BDV genomic RNA with T4 RNA ligase (Life Technologies). .. The RNA was reverse transcribed by using random hexamer primers.

    Construct:

    Article Title: Characterization of phosphorus-regulated miR399 and miR827 and their isomirs in barley under phosphorus-sufficient and phosphorus-deficient conditions
    Article Snippet: Pallas) degradome library constructed using RLM-5'-RACE technology according to Addo-Quaye et al. [ ]. .. Briefly, poly(A) RNA was extracted from total RNA of barley plants using the Oligotex kit (Qiagen) and then ligated with a 5′ RNA adaptor containing a Mme I restriction site using T4 RNA ligase (Invitrogen), followed by reverse transcription, second-strand synthesis, Mm eI digestion, ligation of a 3′ dsDNA adaptor, gel-purification, and PCR amplification.

    Nested PCR:

    Article Title: Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves
    Article Snippet: The cleaved products were uncapped and carried a free phosphate, thereby allowing direct ligation with the RNA adaptor RA44 using T4 RNA Ligase (Ambion, USA). .. Nested PCR was performed using premix ExTaq ™ Hot Start Version (TaKaRa, Dalian, China) and RA44OP/IP and GSP1/GSP2 primers in order to detect the cleaved products.

    Rapid Amplification of cDNA Ends:

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: For 5′ rapid amplification of cDNA ends (RACE), a FirstChoice RLM-RACE kit (Ambion, Austin, TX) was used as recommended by the manufacturer. .. Briefly, 3 to 5 μg of total RNA was ligated to 300 ng of 5′ RACE adaptor using 5 units of T4 RNA ligase (Ambion, Austin, TX) for 1 h at 37°C.

    SDS Page:

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: The purity of the enzymes was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and by western blot using anti-GST antibodies, revealing > 90% homogeneity. .. In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo).

    Plasmid Preparation:

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). .. Templates for in vitro transcription of HIV-2 RNA molecules were obtained by PCR amplification of fragments from the HIV-2 plasmid pROD10-EVA232 using a forward primer containing a T7 promoter sequence (Additional file ).

    Article Title: A Runx2/miR-3960/miR-2861 Regulatory Feedback Loop during Mouse Osteoblast Differentiation *
    Article Snippet: Briefly, small RNAs were polyadenylated, and a 5′-adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase (Invitrogen). .. The PCR products were directly subcloned into pcDNA3.1 TOPO vector (Invitrogen) for sequencing analysis.

    Article Title: Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
    Article Snippet: In order to sequence the 3′ and 5′ ends of the BDV genome, a purified synthetic RNA transcript of known sequence was ligated to the 3′ and 5′ ends of the BDV genomic RNA with T4 RNA ligase (Life Technologies). .. These PCR products were purified and ligated into the pCR 2.1 vector (TA cloning kit; Invitrogen) and sequenced with the Dye Primer cycle-sequencing kit (Applied Biosystems) by using the M13 Reverse and M13 Forward (−40) primer.

    Real-time Polymerase Chain Reaction:

    Article Title: Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
    Article Snippet: All PCRs were performed on either an ABI Prism 7000 real-time PCR detection system (Applied Biosystems, Carlsbad, CA) or an Eppendorf Mastercycler ep realplex 2 apparatus (Eppendorf, Hauppauge, NY) with PerfeCTa SYBR green FastMix, ROX (Quanta Biosciences, Gaithersburg, MD), and gene-specific primers. .. Briefly, 3 to 5 μg of total RNA was ligated to 300 ng of 5′ RACE adaptor using 5 units of T4 RNA ligase (Ambion, Austin, TX) for 1 h at 37°C.

    Agarose Gel Electrophoresis:

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: Immediately after transcription and DNase 1 treatment, the reaction was analysed on a 1% agarose gel as described above. .. For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight.

    In Vitro:

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare). .. Templates for in vitro transcription of HIV-2 RNA molecules were obtained by PCR amplification of fragments from the HIV-2 plasmid pROD10-EVA232 using a forward primer containing a T7 promoter sequence (Additional file ).

    Article Title: DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers
    Article Snippet: .. In vitro activity assays of the proteins were performed using the synthetic 30-mer oligoribonucleotide ss16-A14 as a substrate, labelled at its 5′-end with [γ-32 ATP] and T4 Polynucleotide Kinase (Ambion), and circularized with T4 RNA ligase (Thermo). ..

    Article Title: Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity
    Article Snippet: Paragraph title: In vitro transcription for RNA-RNA interaction assays, RNA-RNA interaction in the presence of ORN and electrophoretic mobility shift assay ... For RNA-ORN hybridization assay, 10pmol of S9 AUG, S9.2, S10 AUG, S10.2, S10.3, S10.5 and Scr ORNs were 3’ end labelled with 10 μCi [32 P]pCp (Perkin Elmer) with T4 RNA ligase (Thermo Scientific) in T4 RNA ligase buffer and incubated at 4°C overnight.

    Ethanol Precipitation:

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: Transcripts were purified by denaturing gel electrophoresis (8 M urea) in 1 × TBE, followed by elution and ethanol precipitation. .. The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare).

    Random Hexamer Labeling:

    Article Title: Enhanced Neurovirulence of Borna Disease Virus Variants Associated with Nucleotide Changes in the Glycoprotein and L Polymerase Genes
    Article Snippet: In order to sequence the 3′ and 5′ ends of the BDV genome, a purified synthetic RNA transcript of known sequence was ligated to the 3′ and 5′ ends of the BDV genomic RNA with T4 RNA ligase (Life Technologies). .. The RNA was reverse transcribed by using random hexamer primers.

    Activation Assay:

    Article Title: The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag
    Article Snippet: DNA and RNA substrates TAR(−) DNA, corresponding to the trans activation response (TAR) sequences of HIV-1MAL , was 32 P-labelled at the 5′-end with [γ-32 P]ATP using T4 polynucleotide kinase (Fermentas) and purified using NucAway Spin Columns (Life Technologies). .. The tRNALys3 was 3′-end labelled using [α-32 P]pCp and T4 RNA ligase (Fermentas) and purified on G50 columns (GE Healthcare).

    Immunoprecipitation:

    Article Title: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis
    Article Snippet: .. Ultra-pure, nuclease and nucleic acid free water (e.g. Milli-Q) 1× Phosphate-Buffered Saline (PBS), RNAse-free (e.g., Invitrogen 10010-023) Tween-20 (e.g., Sigma P9416) Igepal/NP40 substitute (e.g., Sigma I8896) sodium deoxycholate (e.g., Sigma D6750) sodium dodecyl sulfate/SDS (e.g., Sigma L3771) Tris pH 7.5, 1M stock solution (e.g., Sigma 252859) EDTA, 0.5M stock solution (e.g., AM9261) EGTA, 0.5M stock solution (e.g., BioWorld 40520008-2) sodium chloride, 5M stock solution (e.g., Ambion AM9759) potassium chloride, 2M stock solution (e.g., Ambion AM9640G) magnesium chloride, 1M stock solution (e.g., Ambion AM9530G) formamide (e.g., Sigma 47671-250ML-F) ammonium acetate, 1M stock solution (e.g., Sigma A1542) magnesium acetate, 1M stock solution (e.g., Sigma M5661) Dynabeads, Protein A or Protein G-coupled (Invitrogen, 100-01D/100-03D) Bridging antibody: rabbit anti-mouse IgG (only used for Ago CLIP; Jackson ImmunoResearch 315-005-008) Antibody for immunoprecipitation (for Ago CLIP: mouse anti-Ago 2A8, Millipore MABE56) RNase A (molecular biology grade; 20 units/mL) (e.g., Affymetrix/USB, 70194Y) RQ1 DNAse (Promega, M6101) RNasin Plus (Promega, N2611) Alkaline Phosphatase (AP) (Roche, 10713023001) T4 RNA ligase I, 10 units/μL (Fermentas, EL0021, supplied with BSA and 10× buffer) 10 mM ATP (e.g., Thermo Scientific #R0441, diluted 1:10 in water) T4 polynucleotide kinase (PNK), 10 units/μL (NEB, M0201S) [γ-32 P]ATP (3000 Ci/mmol) (Perkin Elmer, BLU002250UC ) Caution: All usage of radioisotopes should be done in strict accordance to the regulations and guidelines of one's institution. ..

    Alkaline Lysis:

    Article Title: Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species
    Article Snippet: We utilized standard alkaline lysis treatment with NaOH-EDTA at 80°C for 15 min to remove any residual RNA, as well as to inactivate the reverse transcriptase and the EXO1 ssDNA nuclease. .. The recovered single stranded cDNA was dried to near completion using a Servo SpeedVac, followed by a second 5′-adenylated linker addition (IDTDNA – Linker 2) to the cDNA using T4 RNA ligase (Ambion).

    Cross-linking Immunoprecipitation:

    Article Title: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis
    Article Snippet: .. Ultra-pure, nuclease and nucleic acid free water (e.g. Milli-Q) 1× Phosphate-Buffered Saline (PBS), RNAse-free (e.g., Invitrogen 10010-023) Tween-20 (e.g., Sigma P9416) Igepal/NP40 substitute (e.g., Sigma I8896) sodium deoxycholate (e.g., Sigma D6750) sodium dodecyl sulfate/SDS (e.g., Sigma L3771) Tris pH 7.5, 1M stock solution (e.g., Sigma 252859) EDTA, 0.5M stock solution (e.g., AM9261) EGTA, 0.5M stock solution (e.g., BioWorld 40520008-2) sodium chloride, 5M stock solution (e.g., Ambion AM9759) potassium chloride, 2M stock solution (e.g., Ambion AM9640G) magnesium chloride, 1M stock solution (e.g., Ambion AM9530G) formamide (e.g., Sigma 47671-250ML-F) ammonium acetate, 1M stock solution (e.g., Sigma A1542) magnesium acetate, 1M stock solution (e.g., Sigma M5661) Dynabeads, Protein A or Protein G-coupled (Invitrogen, 100-01D/100-03D) Bridging antibody: rabbit anti-mouse IgG (only used for Ago CLIP; Jackson ImmunoResearch 315-005-008) Antibody for immunoprecipitation (for Ago CLIP: mouse anti-Ago 2A8, Millipore MABE56) RNase A (molecular biology grade; 20 units/mL) (e.g., Affymetrix/USB, 70194Y) RQ1 DNAse (Promega, M6101) RNasin Plus (Promega, N2611) Alkaline Phosphatase (AP) (Roche, 10713023001) T4 RNA ligase I, 10 units/μL (Fermentas, EL0021, supplied with BSA and 10× buffer) 10 mM ATP (e.g., Thermo Scientific #R0441, diluted 1:10 in water) T4 polynucleotide kinase (PNK), 10 units/μL (NEB, M0201S) [γ-32 P]ATP (3000 Ci/mmol) (Perkin Elmer, BLU002250UC ) Caution: All usage of radioisotopes should be done in strict accordance to the regulations and guidelines of one's institution. ..

    other:

    Article Title: RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1) *
    Article Snippet: RNAs were added to a 50-μl reaction containing 50 m m Tris/Tris-HCl (pH 7.5), 10 m m MgCl2 , 10 m m DTT, 1 m m ATP, 10% PEG, 100 μg/ml BSA, 50 units of T4 RNA ligase, 3 μ m RNA, and 30 μ m pCp-Biotin.

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  • 90
    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: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/Thermo Fisher
    Average 90 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    90
    Thermo Fisher t4 dna ligation 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 Ligation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligation buffer/product/Thermo Fisher
    Average 90 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligation buffer - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

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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

    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 5΄-end ligation; ss2 (final conc 10 μM), T4 DNA ligation buffer, PEG4000 (5%, w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free H2 O was added to the sample on ice, in a total volume of 20 μl.

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