T4 polynucleotide kinase t4 pnk

Manufactured by New England Biolabs

Sourced in United States

T4 polynucleotide kinase (T4 PNK) is an enzyme that catalyzes the transfer of a phosphate group from ATP to the 5' hydroxyl terminus of DNA, RNA, or oligonucleotides. This enzyme is commonly used in molecular biology applications to label the 5' end of nucleic acid molecules.

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Lab products found in correlation

γ 32p atp, by PerkinElmer (4 mentions) T4 dna ligase, by New England Biolabs (4 mentions) Phusion hf polymerase, by New England Biolabs (2 mentions) Kapa hifi dna polymerase, by Roche (1 mentions) Nebnext magnesium rna fragmentation module, by New England Biolabs (1 mentions) Small rna sample preparation kit, by New England Biolabs (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) Abi 394 dna synthesizer, by Thermo Fisher Scientific (1 mentions) Ac dc ce, by Glen Research (1 mentions) Dt ce, by Glen Research (1 mentions) Illustra nap 5 desalting columns, by GE Healthcare (1 mentions) Micro bio spin 6 column, by Bio-Rad (1 mentions) T4 dna ligase, by Roche (1 mentions) Biospin columns, by Bio-Rad (1 mentions) E coli uracil dna glycosylase udg, by New England Biolabs (1 mentions) Human pol β, by Bio-Techne (1 mentions) Acrylamide bis acrylamide solution, by Bio-Rad (1 mentions) Gibson cloning mastermix, by New England Biolabs (1 mentions) Snapgene software, by GSL Biotech (1 mentions) Calf intestinal alkaline phosphatase, by New England Biolabs (1 mentions)

18 protocols using t4 polynucleotide kinase t4 pnk

Mutagenesis of BIM splicing regulators

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We made mutant plasmids in the context of both BIM Δ10 and Δ11 minigenes [31 (link)], using PCR mutagenesis with specific primers (sequences available upon request) and KAPA HiFi DNA polymerase (KAPA Biosystems). We generated serial deletions and staggered deletions of 10 nucleotides each in BIM E3 and upstream 106 nt intronic region, by using specific primers which contained the flanking regions of the deleted sequence. We introduced point mutations in enhancer sequences of BIM E3 using specific primers. pCGT7-empty and pCGT7-SRSF1 plasmids were gifted by Prof Javier F Cáceres from MRC Human Genetics Unit at Edinburgh, UK [65 (link)].
The pSXN plasmidwas provided by Prof Thomas A Cooper from Baylor College of Medicine, USA [64 (link)]. We annealed equimolar amounts of a pair of complementary DNA oligonucleotides containing designed test sequences and restriction overhangs by heating at 95°C for 5 min followed by gradual cooling to room temperature for 1 h. We then 5′-end phosphorylated the annealed oligonucleotides by T4 polynucleotide kinase (T4PNK; New England Biolabs) in 1x ligation buffer, and ligated the duplex into the BamHI and SalI sites in the alternative exon within pSXN13 vector.

Liu J., Bhadra M., Sinnakannu J.R., Yue W.L., Tan C.W., Rigo F., Ong S.T, & Roca X. (2017). Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides. Oncotarget, 8(44), 77567-77585.

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Transcriptome and Genome Profiling of Rice

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About 1 μg genomic DNA from the R and S plants were used for DNA library preparation, using the SOLiD™ 5500 Fragment Library Core Kit (Part no. 4464412) according to its user guide. A total of 20 μg total RNA was used for two rounds of mRNA purification using Dynabeads (610.06, Invitrogen, USA). About 100 ng mRNA was fragmented using NEB Next Magnesium RNA Fragmentation Module (E6150, NEB), purified with an RNA clean up kit (R6247, Omega, USA), end repaired with T4 Polynucleotide Kinase (T4 PNK) (M0201, NEB) and cleaned up again with a kit (R6247, Omega). The end-repaired RNAs were used to prepare the strand specific transcriptome, using the Small RNA Sample Preparation kit (E6160, NEB) according to the manufacturer protocol with some minor modification, including the SR Primer F3 being replaced with barcode primers. The resulting DNA and RNA libraries were used for emulsion PCR to produce the beads for sequencing on the SOLiD 5500 machine, using 75 nt mode and 75 nt + 35 nt mode for the sequencing of DNA and RNA libraries, respectively. Biological duplicates of RNA libraries of the R and S plants before Xoo infections were profiled for quality assessment.

Peng H., Chen Z., Fang Z., Zhou J., Xia Z., Gao L., Chen L., Li L., Li T., Zhai W, & Zhang W. (2015). Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection. Scientific Reports, 5, 12165.

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Synthesis and Characterization of Modified DNA Oligonucleotides

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Protected 2′-deoxyribonucleoside-3′-phosphoramidites (PAC-dA-CE, Ac-dC-CE, p-iPr-PAC-dG-CE, dT-CE, 8-oxo-dG-CE, 1,N⁶-etheno-dA-CE, 5-fluoro-dC-CE) Ac-dC-CPG ABI and p-iPr-PAC-dG-CPG ABI columns, and all other reagents necessary for automated DNA synthesis were purchased from Glen Research (Sterling, VA). 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl)-N,N-diisopropyl-phosphoramidite of 6-chloropurine-2′-deoxyriboside was purchased from ChemGenes Corp. (Wilmington, MA). Synthetic DNA oligodeoxynucleotides were prepared by solid phase synthesis using an ABI 394 DNA synthesizer (Applied Biosystems, CA). DNA oligodeoxynucleotides containing 8-oxo-dG and thymine glycol were purchased from Integrated DNA Technologies (Coralville, IA) and Sigma Aldrich (St. Louis, MO), respectively. T4 polynucleotide kinase (T4-PNK) was obtained from New England Biolabs (Beverly, MA), while T4 DNA ligase was procured from Roche (Basel, Switzerland). γ-³²P ATP was purchased from Perkin-Elmer Life Sciences (Boston, MA). 40% 19:1 acrylamide/bis solution and micro bio-spin 6 columns were purchased from Bio-Rad (Hercules, CA). Illustra NAP-5 desalting columns and Sep-Pak C18 SPE cartridges were obtained from GE Healthcare (Pittsburg, PA) and Waters (Milford, MA), respectively. All other chemicals and solvents were purchased from Sigma-Aldrich (Milwaukee, WI) and used without further purification.

Wickramaratne S., Banda D., Ji S., Manlove A.H., Malayappan B., Nuñez N.N., Samson L., Campbell C., David S, & Tretyakova N. (2016). Base Excision Repair of N6-Deoxyadenosine Adducts of 1,3-Butadiene. Biochemistry, 55(43), 6070-6081.

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Recombinant Human Polymerases for Gel Electrophoresis

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Full-length recombinant human polymerase κ (hPol κ) used for gel electrophoresis experiments was purchased from Enzymax (Lexington, KY). Human Pol β was obtained from Trevigen (Gaithersburg, MD). Recombinant human DNA polymerases hPol η (amino acids 1–437), hPol ι (amino acids 1–420), and hPol κ (amino acids 19–526) (active core enzymes) were expressed and purified according to previously published procedures.^{44 (link)–46 (link)} T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA). [γ-³²P]ATP was purchased from Perkin-Elmer Life Sciences (Boston, MA). Acrylamide/bis-acrylamide solution (40% 19:1, w/w) and Bio-spin columns were obtained from Bio-Rad laboratories (Hercules, CA). All other chemicals were purchased either from Sigma-Aldrich or Fisher Scientific.

Kotapati S., Wickramaratne S., Esades A., Boldry E.J., Dorr D.Q., Pence M.G., Guengerich F.P, & Tretyakova N.Y. (2015). Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene. Chemical research in toxicology, 28(7), 1496-1507.

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Mutation of ADAM10 by Gibson Assembly

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Mutation of the identified insertion TEVcs was introduced on the pRK5M-ADAM10 plasmid (addgene: 31717) through the Gibson Assembly reaction (Gibson Cloning MasterMix, NEB, Ipswich, MA, USA) following the manufacturer’s instructions. All the primers were designed with by SnapGene software (GSL Biotech, San Diego, CA, USA). The insertion of the T719A point mutation was introduced by PCR extension and ligation with T4 Polynucleotide Kinase (T4 PNK, NEB, Ipswich, MA, USA) and T4 DNA Ligase (NEB, Ipswich, MA, USA). Protein structure modeling was performed using AlphaFold2 as previously described [63 (link)]. Sequencing data were inspected using Sanger Sequencing and Fragment Analysis Software SeqScape of Applied Biosystems (ThermoFisher Scientific, Waltham, MA, USA) and assisted by SnapGene software (GSL Biotech, San Diego, CA, USA).

Pastore F., Battistoni M., Sollazzo R., Renna P., Paciello F., Li Puma D.D., Barone E., Dagliyan O., Ripoli C, & Grassi C. (2023). A Bioengineering Strategy to Control ADAM10 Activity in Living Cells. International Journal of Molecular Sciences, 24(2), 917.

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Enzymatic Modifications of Oligonucleotides

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All of the unmodified oligos were synthesized and purified [by high performance liquid chromatography (HPLC)] by Sangon Biological Engineering Technology and Services (Shanghai, China). T4 DNA ligase, T4 polynucleotide kinase (T4 PNK), calf intestinal alkaline phosphatase, deoxyribonuclease I and adenosine triphosphate (ATP) were purchased from New England BioLabs (Ipswich, MA, USA). Snake venom phosphodiesterase, boronic acid (BA), phenylboronic acid (PBA), 3-chlorophenylboronic acid (3-CPBA), 2-(2′-chlorobenzyloxy) phenylboronic acid (2-CB-PBA) and 3-(Dansylamino) phenylboronic acid (3-D-PBA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5mdCTP and 5hmdCTP were purchased from Zymo Research (Irvine, CA, USA), and deoxyguanosine triphosphate (dGTP), thymine triphosphate (TTP), deoxycytidine (dCTP), GO Taq hot start polymerase were ordered from Promega (Madison, WI, USA). Other biochemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). The 5hmC-ds83mers with a sequence of 5′-TTTCCTACCT TAAGATCCTT CCAGTCTC CGCCGCG CAGTG TTACCCTTAG AGCTCATACC ATTCGCCAAT TTCTTCGCAC GTT-3′ (only one strand shown) were synthesized and purified by TaKaRa Biotechnology Co., Ltd. (Dalian, China).

Zhao C., Wang H., Zhao B., Li C., Yin R., Song M., Liu B., Liu Z, & Jiang G. (2014). Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine. Nucleic Acids Research, 42(9), e81.

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Spike-in RNA Sequencing for Cellular and Extracellular Transcripts

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Two sets of spike-in RNAs were added to the samples prior to library preparation: ERCC RNA Spike-In Control Mixes (Ambion; 0.02 μl per 1 μg total RNA), and miRCURY Spike-in kit, part 1, with UniSp2’s final concentrations of 1.25 and 5.0 fmol per 1 μg of total RNA for cellular and extracellular RNA, respectively. Total RNA, either 40–200 ng of exRNA, or 2 μg of cellular RNA, was rRNA-depleted using the Ribo-Zero rRNA Removal Kits (Illumina, CA). One quarter of the rRNA-depleted RNA was fragmented to 100–500 nt using the 5× First-Strand Buffer (Clontech, CA), and utilized for the long RNA library construction by SMARTer Stranded RNA-Seq Kit (Clontech). The remaining 75% of the rRNA-depleted RNA was treated sequentially with Tobacco Acid Pyrophosphatase (TAP; Illumina) and T4 Polynucleotide Kinase (T4PNK; New England Biolabs, MA) to create more uniform 5′- and 3′-ends for various classes of transcripts. The RNA was then used as input for the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs), with size selection of 15–65 nt inserts for small RNA libraries. The quality of libraries was examined using the Agilent DNA 1000 kit at the Agilent 2100 Bioanalyzer instrument, and cDNA quantified by qRT-PCR. The libraries were sequenced on HiSeq 2000 (Illumina) with single read 50 cycles by Beijing Genomics Institute (BGI, China).

Wei Z., Batagov A.O., Schinelli S., Wang J., Wang Y., El Fatimy R., Rabinovsky R., Balaj L., Chen C.C., Hochberg F., Carter B., Breakefield X.O, & Krichevsky A.M. (2017). Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nature Communications, 8, 1145.

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Biochemical Assay for Pyrophosphate Detection

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Sodium alginate, tryptophan, and pyrophosphate (PPi) were obtained from Aladdin reagent Co., Ltd. (Shanghai, China). Alkaline phosphatse (ALP) from calf intestinal mucosa, T4 Polynucleotide Kinase (T4 PNK), and exonuclease III (Exo III) were purchased from New England biolabs (Ipswich, MA). Adenosine triphosphate (ATP), bovine serum albumin (BSA), glucose oxidase (GOx), and thrombin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents of analytical grade were directly used without additional purification. The reaction buffer solution employed in this work was 10 mM Tris-HCl (pH 7.4). All solutions were prepared by using ultrapure water which was obtained through a Millipore Milli-Q water purification system (Billerica, MA) with an electric resistance ≥18.2 MΩ.

Hu Y., Geng X., Zhang L., Huang Z., Ge J, & Li Z. (2017). Nitrogen-doped Carbon Dots Mediated Fluorescent on-off Assay for Rapid and Highly Sensitive Pyrophosphate and Alkaline Phosphatase Detection. Scientific Reports, 7, 5849.

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Thrombin Aptamers for Anticoagulant Activity

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Thrombin and phosphate-buffered saline (PBS) were purchased from Solarbio Science and Technology Co., Ltd., Beijing, China. Fibrinogen (FIB), lysozyme (Lys), and cytochrome C (Cyt C) were purchased from Sangon Biotech Co., Ltd., Shanghai, China. T4 polynucleotide kinase (T4 PNK) was supplied by New England Biolabs Inc., Ipswich, MA, USA. 4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate (SITS) was purchased from Sigma Aldrich, Shanghai, China. Thrombin aptamers (TA) (Thrombin15, Thrombin27, and Thrombin29) which were NH₂-modified at different sites were synthesized by Sangon Biotech Co., Ltd., Shanghai, China (see Table S1). Chromatographic-grade acetonitrile was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Sodium carbonate (Na₂CO₃), sodium bicarbonate (NaHCO₃), sodium chloride (NaCl), and ammonium acetate were commercially available analytical reagents supplied by Damao Chemical Reagent Factory, Tianjin, China. The photosensitive materials were protected from light during operation.

Zeng X., Zhou Q., Wang L., Zhu X., Cui K., Peng X., Steele T.W., Chen H., Xu H, & Zhou Y. (2021). A Fluorescence Kinetic-Based Aptasensor Employing Stilbene Isomerization for Detection of Thrombin. Materials, 14(22), 6927.

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Radioactive Oligonucleotide Biochemical Assays

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Oligonucleotides used in biochemical assays were synthesized by BGI, Shenzhen, China, and their sequences are listed in Supplementary Table S1. Radioactive oligonucleotides were labeled with [γ-³²P] ATP (6000 Ci/mmol) (PerkinElmer Life Sciences) using T4 polynucleotide kinase (T4 PNK) (New England Biolabs). The SSB-coated primed-M13mp18 ssDNA template used in the leading-strand replication assay was prepared by annealing a 5′-³²P-labeled 30-mer (map positon 6852–6881) to M13mp18 ssDNA (New England Biolabs) in a 1:1 molar ratio in annealing buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl) at 94 °C for 4 min and then slowly cooling it to room temperature over 8 h. The annealed DNA mixture was then incubated with a 350-fold molar excess of SSB₄ at 16 °C for 2 h. In exonuclease activity assays, 5′-³²P-labeled 40-mers or 5′-³²P-labeled 20-mers were adopted as single-strand DNA substrates, and 5′-³²P-labeled 40-mers were annealed to the unlabeled single-stranded oligonucleotides Template-40 (40-mers) and Template-50 (50-mers) in a 1:1.2 molar ratio to produce radioactive blunt-dsDNA and 5′ overhanging-dsDNA substrates, respectively. In primer-extension assays, a 5′-³²P-labeled 20-mer was annealed to unlabeled ssDNA Template-40 to serve as the substrate.

Gu S., Li W., Zhang H., Fleming J., Yang W., Wang S., Wei W., Zhou J., Zhu G., Deng J., Hou J., Zhou Y., Lin S., Zhang X.E, & Bi L. (2016). The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity. Scientific Reports, 6, 18418.

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