T4 rna ligase reaction buffer

Manufactured by New England Biolabs

T4 RNA ligase reaction buffer is a solution designed for use in enzymatic reactions involving T4 RNA ligase. It provides the optimal ionic conditions and cofactors required for the enzyme's activity. The buffer composition supports the ligation of RNA molecules.

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Lab products found in correlation

T4 rna ligase 1, by New England Biolabs (4 mentions) Peg8000, by New England Biolabs (3 mentions) Ribolock rnase inhibitor, by Thermo Fisher Scientific (2 mentions) Myone streptavidin c1 magnetic beads, by Thermo Fisher Scientific (2 mentions) T3 dna ligase, by New England Biolabs (2 mentions) Adenosine triphosphate (atp), by New England Biolabs (1 mentions) T4 pnk reaction buffer, by New England Biolabs (1 mentions) Rnase inhibitor, by New England Biolabs (1 mentions) T4 pnk, by New England Biolabs (1 mentions) Pcp cy5, by Jena Biosciences (1 mentions) Reverse transcriptase m mlv rnase h, by Takara Bio (1 mentions) Rneasy minelute cleanup kit, by Qiagen (1 mentions) Pgem t easy vector, by Promega (1 mentions)

8 protocols using t4 rna ligase reaction buffer

RNA Adapter Ligation Protocol

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Purified RNA (10.7 μl) was mixed with 2 μl of T4 RNA Ligase Reaction buffer (New England Biolabs), 1 μl of 30 μM RA3 adapter oligonucleotide, 4.8 μl of 50% PEG 8000 (New England Biolabs) and 0.5 μl of RiboLock RNase inhibitor (40 U/μl; Thermo Fisher Scientific). After 2 min of incubation at 70°C, the tube was placed on ice and 1 μl of T4 RNA Ligase 2, Truncated (200 U/μl; New England Biolabs) was added. The reaction was performed for 1 h at 25°C.

Malik D., Kobyłecki K., Krawczyk P., Poznański J., Jakielaszek A., Napiórkowska A., Dziembowski A., Tomecki R, & Nowotny M. (2020). Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine. Nucleic Acids Research, 48(16), 9387-9405.

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Ligating T7 Promoter to Precursor 1

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To ligate the complementary sequence of the T7 promoter to the 3′-end of precursor 1, 1 pmol of precursor 1 and 100 pmol of a 5′-phosphorylated ss oligonucleotide corresponding to the reverse complement of the T7 promoter (T7 linker) were mixed in a 20 µL volume containing 1× T4 RNA Ligase reaction buffer (NEB), 1 mM ATP (NEB), 25% (w/v) PEG 8000 (NEB), and 10 units of T4 RNA Ligase 1 (NEB). To prevent formation of concatemers, the T7 linker contained a 3′ chain terminator (2′,3′-dideoxycytosine). The reaction and negative control were incubated at 22°C overnight. Ligation of the two oligonucleotides was confirmed by gel electrophoresis (Supplemental Fig. S8). Ligation reaction was ethanol precipitated and the product (precursor 1-T7) was gel-purified using TE buffer.

Döring J, & Hurek T. (2019). Dual coding potential of a 2′,5′-branched ribonucleotide in DNA. RNA, 25(1), 105-120.

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RNA Adapter Ligation Protocol

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Of note, 10.7 µL of purified RNA was mixed with 2 µL of T4 RNA Ligase Reaction buffer (New England Biolabs), 1 µL of 30 µM RA3 adapter, 4.8 µL of 50% PEG 8000 (New England Biolabs) and 0.5 µL of RiboLock RNase Inhibitor (40 U/µL; Thermo Fisher Scientific). After 2 min of incubation at 70°C, the tube was placed on ice, and 1 µL of T4 RNA Ligase 2, Truncated (200 U/µL; New England Biolabs) was added. The reaction was carried out for 1 h at 25°C.

Kobyłecki K., Kuchta K., Dziembowski A., Ginalski K, & Tomecki R. (2017). Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3′-terminal positions of synthesized tails. RNA, 23(12), 1902-1926.

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Enzymatic Synthesis of Customized DNA

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DNA template [up to 10 pmol, either in solution or immobilized on MyOne Streptavidin C1 magnetic beads (ThermoFisher Scientific)], polymerization initiation and termination primers (1.5 equivalents each relative to template), functionalized trinucleotide building blocks (10 equivalents relative to template for each occurrence of the corresponding codon) and 10x T4 RNA ligase reaction buffer (New England Biolabs; 1 μL) were mixed in a total volume of 8 μL in a PCR tube. The mixture was subjected to the following temperature program on a thermocycler: 95 °C for 10 sec; 65 °C for 4 min; a ramp from 65 °C to 4 °C at 0.1 °C per 10 s. To the PCR tube were added 1 μL of 10 mM ATP and 1 μL of T3 DNA ligase (New England Biolabs). The reaction was incubated at 4 °C for 12 h and then at 16 °C for 2 h.

Chen Z., Lichtor P.A., Berliner A.P., Chen J.C, & Liu D.R. (2018). Evolution of Sequence-Defined Highly Functionalized Nucleic Acid Polymers. Nature chemistry, 10(4), 420-427.

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Enzymatic Synthesis of Customized DNA

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Chen Z., Lichtor P.A., Berliner A.P., Chen J.C, & Liu D.R. (2018). Evolution of Sequence-Defined Highly Functionalized Nucleic Acid Polymers. Nature chemistry, 10(4), 420-427.

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Reducing RNA Fragmentation Background in Pseudouridine Mapping

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The CMC reaction and the reversal steps can cause RNA fragmentation, resulting in elevated background signals and false-positive results. In order to reduce background signal at target sites, a “blocking” RNA ligation step is added in our method. The ligated 5-mer RNA oligonucleotide interrupts the splint ligation by fragmented RNA at target Ψ site and reduces background. Ligation of this blocking 5-mer RNA requires a 5’ phosphate of the CMC-induced fragment; therefore 5’ phosphorylation is first carried out before ligation.

To 6 μg +CMC treated or 4 μg −CMC treated total RNA in 6.5 μl, add 1μl 10X T4 PNK reaction buffer (B0201S, NEB), 1 μl 1 mM ATP, 0.5 μl RNase inhibitor (M0307L, NEB), and 1 μl 10 U/μl T4 PNK (M0201L, NEB) to each tube and mix.

Incubate samples at 37°C for 30 min, followed by a quick spin.

Add 1 μl 10×T4 RNA Ligase Reaction Buffer (B0216L, NEB), 1 μl 100 μM 5’ RNA blocker oligo (/5AmMC6/rArCrCrCrA, IDT), 1 μl 1 mM ATP, 1 μl RNase inhibitor, 3 μl DMSO, 2 μl sterile water, and 1 μl 10 U/μl T4 RNA ligase I (M0204L, NEB).

Incubate the reaction mixtures at 16°C for 16 hours.

Add 1.2 μl 200 mM EDTA to each tube to stop the reaction.

Zhang W, & Pan T. (2021). Pseudouridine RNA modification detection and quantification by RT-PCR. Methods (San Diego, Calif.), 203, 1-4.

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RNase E UTR Labeling with pCp Cy5

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In vitro transcribed RNase E 5’ UTR (4.5 μM) was incubated with 33 μM of pCp Cy5 (Jena Bioscience #NU-1706-CY5) in a total reaction volume of 100 μL consisting of 50 units of T4 RNA ligase 1 (NEB #M0204S), 1mM ATP, 10% DMSO1, 1X T4 RNA ligase reaction buffer (NEB #B0216S) for 16 hours at 16°C. Following the reaction, T4 RNA ligase 1 was heat inactivated at 65°C for 15 minutes and the reaction mixture was subjected to Phenol-Chloroform extraction to remove the enzyme. The unincorporated pCp Cy5 was removed by passing the labeled RNA through Sephadex G-50 column (Cytiva # 28903408) and eluted in nuclease free water.

Nandana V., Al-Husini N., Vaishnav A., Dilrangi K.H, & Schrader J.M. (2023). Caulobacter crescentus RNase E condensation contributes to autoregulation and fitness. bioRxiv.

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Inverted RT-PCR for Circularized RNA

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Inverted RT-PCR was performed on circularized total RNA extracted from 5 days post-fertilisation (dpf) WT and rcl1^hi2452 mutant embryos, as described previously (32 (link)). Briefly, 5 μg total RNA and T4 RNA Ligase Reaction Buffer (New England Biolabs, M0204S) in a total volume of 15 μl were first incubated for 4 min at 75°C, followed by rapid freezing in pre-cooled ethanol at −80°C for 1 min. Frozen samples were then placed on ice to thaw slowly before PEG8000 was added. Subsequently, ATP, RNase inhibitor (New England Biolabs, M0314) and 2μl T4 RNA Ligase 1 (New England Biolabs, M0204S) were added to yield a final reaction volume of 20 μl according to the manufacturer's instructions. The reaction mixture was incubated at 25°C for 2 h and then boiled for 2 min to terminate the reaction. The circularized total RNA was purified using RNeasy MinElute Cleanup Kit (QIAGEN, 74204) and then subjected to reverse transcription (RT) using Reverse Transcriptase M-MLV (RNase H-) (Takara, 2641A) with random primers. The cDNA was used as the template for RT-PCR with the primer pairs 18S-R1 and 18S-F1 (P1), 18S-R1 and 18S-F2 (P2), 18S-R2 and 18S-F1 (P3), and 18S-R1 and 28S-F (P0) (Supplementary Table S1). The PCR products were ligated into the pGEM-T Easy Vector (Promega, A1360) and single colonies were picked for sequencing.

Zhu Q., Tao B., Chen H., Shi H., Huang L., Chen J., Hu M., Lo L.J, & Peng J. (2021). Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and up-regulates a cohort of ribosome biogenesis genes in zebrafish. Nucleic Acids Research, 49(10), 5743-5759.

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