T4 pnk reaction buffer

Manufactured by New England Biolabs

T4 PNK reaction buffer is a solution used in molecular biology protocols that require the activity of T4 polynucleotide kinase (T4 PNK). T4 PNK is an enzyme that catalyzes the transfer of a phosphate group from ATP to the 5' end of DNA or RNA molecules, enabling their subsequent use in various downstream applications.

Automatically generated - may contain errors

Lab products found in correlation

T4 pnk, by New England Biolabs (4 mentions) Rnase inhibitor, by New England Biolabs (1 mentions) T4 rna ligase 1, by New England Biolabs (1 mentions) T4 rna ligase reaction buffer, by New England Biolabs (1 mentions) Thermomixer, by Eppendorf (1 mentions) Rna clean concentrator kit, by Zymo Research (1 mentions) Nuclease free water, by Thermo Fisher Scientific (1 mentions) Adenosine triphosphate (atp), by New England Biolabs (1 mentions) Turbo dna free kit, by Thermo Fisher Scientific (1 mentions) Cap clip acid pyrophosphatase, by CellScript (1 mentions)

Spelling variants of this product

T4 PNK (345 citations) PNK buffer (22 citations) T4 PNK buffer (14 citations) Reaction buffer (11 citations) PNK reaction buffer (5 citations)

4 protocols using t4 pnk reaction buffer

Reducing RNA Fragmentation Background in Pseudouridine Mapping

Check if the same lab product or an alternative is used in the 5 most similar protocols

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.

+ Open protocol

+ Expand

Oligonucleotide Isolation and Radiolabeling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Individual oligonucleotides for helicase template preparation (Table 3) were gel-isolated using the crush and soak method: Oligonucleotides were electrophoresed on 8% denaturing polyacrylamide gels at 120 V for 1.5 hr in 1× TBE buffer. The bands were excised, crushed into a fine paste, and soaked overnight in an Eppendorf ThermoMixer at 37°C and 1400 rpm in 500 mM ammonium acetate/10 mM magnesium acetate/1 mM EDTA. The extracted oligonucleotides were ethanol-precipitated twice and stored in TE pH 8.0.
Radiolabeling of oligonucleotides was carried out for 1 hr at 37°C using T4 PNK (NEB), 0.25 μM oligonucleotide, and 0.5 μM γ-[³²P]-ATP. Reactions were terminated by incubation for 20 min at 80°C. Following the radiolabeling, oligonucleotide annealing was carried out in a thermocycler in T4 PNK reaction buffer (NEB)/50 mM potassium acetate by heating oligonucleotide mixtures to 95°C for 5 min, followed by cooling at a rate of 1°C/min until the temperature reached 10°C. Annealed products were fractionated by 10% native PAGE at 150 V for 45 min in 0.5× TBE, corresponding bands excised from the gel, and templates eluted by soaking the excised gel slices overnight in buffer containing 10 mM Tris-HCl pH 7.2 (for templates with RNA) or pH 8.0 (for templates with DNA)/50 mM potassium acetate/1 mM EDTA.

Kumar C., Batra S., Griffith J.D, & Remus D. (2021). The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions. eLife, 10, e72286.

+ Open protocol

+ Expand

FFPE RNA Dephosphorylation and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols

FFPE Total RNA (250 ng) was dissolved in nuclease-free water (up to final volume of 50 μl), followed by 10× T4 PNK Reaction Buffer (NEB #B0201), ATP (final concentration 1 mM; NEB #P0756), and T4 PNK (1 μl; NEB #M0201). The reaction was incubated for 30 min at 37°C after which time the material was subjected to purification using a Zymo RNA Clean & Concentrator Kit following the standard protocol.

Wulf M.G., Maguire S., Dai N., Blondel A., Posfai D., Krishnan K., Sun Z., Guan S, & Corrêa IR J.r. (2021). Chemical capping improves template switching and enhances sequencing of small RNAs. Nucleic Acids Research, 50(1), e2.

+ Open protocol

+ Expand

Small RNA Deacylation and Demethylation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Two micrograms of small RNA from each sample was incubated with deacylation buffer (pH = 9.0) at 37 °C for 45 min and followed by ethanol precipitation. The small RNA was recovered in 20 µL nuclease-free water (Ambion). Then the recovered small RNA was treated with TURBO DNA-free kit (Ambion) for DNA contamination removing. The small RNA was purified from the reaction by ethanol precipitation. Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min. The small RNA was purified from the reaction by phenol-chloroform extraction and ethanol precipitation. Then the purified small RNA was treated with 2× molar ratio of AlkB and 4× molar ratio of AlkB (D135S) at 25 °C for 1 h with 300 mM KCl, 2 mM MgCl₂, 10 µM of (NH4)2Fe(SO4)2·6H₂O, 300 µM 2-ketoglutarate (2-KG), 2 mM L-ascorbic acid, 50 µg/ml BSA, 50 mM MES buffer (pH 5.0). The reaction was quenched by addition of 5 mM EDTA. After phenol-chloroform extraction and ethanol precipitation, the small RNA was recovered in 3 µL nuclease-free water (Ambion).

Wang H., Huang R., Li L., Zhu J., Li Z., Peng C., Zhuang X., Lin H., Shi S, & Huang P. (2021). CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini. Cell Discovery, 7, 25.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!