5′-end labeling of substrates was performed using [γ-32P] adenosine triphosphate (Hartmann Analytic) with T4 polynucleotide kinase (NEB). After labeling, oligonucleotides were subjected to phenol–chloroform extraction, precipitated, and purified by denaturing polyacrylamide gel electrophoresis.
T4 polynucleotide kinase
Manufactured by Hartmann Analytic
The T4 polynucleotide kinase is an enzyme used in molecular biology to catalyze the transfer of a phosphate group from ATP to the 5' hydroxyl terminus of DNA, RNA, or oligonucleotides. This process is commonly used in various DNA manipulation and analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
γ 32p atp, by Hartmann Analytic (5 mentions)
G 25 column, by GE Healthcare (1 mentions)
Qubit protein assay, by Thermo Fisher Scientific (1 mentions)
Hybond n membrane, by Cytiva (1 mentions)
Hybond, by Cytiva (1 mentions)
α32p atp, by Hartmann Analytic (1 mentions)
E coli mre 600, by Roche (1 mentions)
Typhoon 9410 variable imager, by GE Healthcare (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
Glycoblue coprecipitant, by Thermo Fisher Scientific (1 mentions)
6 protocols using t4 polynucleotide kinase
1
Radioactive Labeling of Oligonucleotides
2
Radiolabeling RNA Oligonucleotides for Analysis
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In vitro synthesized RNA oligonucleotides were diluted to 250 nM with nuclease-free water and mixed with T4 polynucleotide kinase buffer. The RNA was refolded by heating the mixture at 95°C for 3 min and snap-cooled on ice for 5 min. After addition of RNase inhibitors (NEB), T4 polynucleotide kinase (NEB), and [γ-32P]-ATP (HARTMANN ANALYTIC), the reaction was incubated at 37°C for 10 minutes. The 5′-radiolabelled RNA was purified on G-25 columns (GE Healthcare) and diluted to a final concentration of 50 nM. The radiolabelled RNA Decade Marker (ThermoFisher Scientific) was prepared according to the manual. The RNA and the marker were aliquoted and stored at -20°C.
3
Quantitative Assay of TOP3A Variants
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The concentrations of purified TOP3A variants were measured using a Qubit protein assay (Thermo Fisher Scientific). Reactions were carried out in a total volume of 20 μl in reaction buffer (25 mM Tris–HCl pH 7.4, 5 mM MgCl2, 50 mM NaCl, 1 mM DTT and 100 μg/ml BSA) at 37°C for 30 min. Proteins were diluted using dilution buffer (20 mM tris‐Cl pH 8, 200 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 0.5 mM EDTA and 100 μg/ml BSA). In experiments involving two compound heterozygous variants, reactions contained 50 fmol of protein (for single variants) or 25 fmol for each of the two variants. The reaction products were then separated by denaturing PAGE as described above. Reaction products were electroblotted onto Hybond‐N+ membranes (Cytiva) at 50 V in 1 × TBE for 1 h. Membranes were crosslinked by exposure to 1200 mJ/cm2 of 254 nm UV. Membranes were hybridised overnight at 42°C with 5′ radioactively labelled DNA probes complementary to R1 or R2 (see Appendix Table S1 for probe sequences). For probe synthesis, 10 pmol of each of R1 probe and R2 probe were labelled using T4 polynucleotide kinase (NEB) and 2 μl of [γ‐32P] ATP (10 mCi/ml, 3,000 Ci/mmol, Hartmann Analytic). The following day, membranes were washed for 3 × 20 min with saline‐sodium citrate (SSC) buffer containing 0.1% SDS and imaged using a Typhoon FLA 9500.
4
Ribozyme Cleavage Fragment Generation
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Ribozyme cleavage fragments (Supplementary Table S2) were generated by T7-based in vitro transcription from PCR products (with T7 promoter) of the respective ribozyme (Supplementary Table S5) [65 (link)] in the presence of [α32P]-ATP (Hartmann Analytic). The cleavage fragments were gel-purified as described previously [66 ]. 3ʹ-DNA adapter
(5ʹ-TGGAATTCTCGGGTGCCAAGG-Amino-C7-3ʹ), RT primer (5′-GCCTTGGCACCCGAGAATTCCA-3′) and circularization-RT primer (5′-P-GATCGTCGGACTGTAGAACTCTGAAC /iSp18/ CACTCA/ iSP18/ GCCTTGGCACCCGAGAATTCCA-3′) (Supplementary Table S5) were radioactively labelled using [γ32P]-ATP (Hartmann Analytic) and T4 Polynucleotide kinase (NEB).
(5ʹ-TGGAATTCTCGGGTGCCAAGG-Amino-C7-3ʹ), RT primer (5′-GCCTTGGCACCCGAGAATTCCA-3′) and circularization-RT primer (5′-P-GATCGTCGGACTGTAGAACTCTGAAC /iSp18/ CACTCA/ iSP18/ GCCTTGGCACCCGAGAATTCCA-3′) (Supplementary Table S5) were radioactively labelled using [γ32P]-ATP (Hartmann Analytic) and T4 Polynucleotide kinase (NEB).
5
Streptomycin Aptamer Structural Analysis
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Streptomycin aptamer and Strep-RS7 RNAs were generated by in vitro transcription with T7 RNA polymerase and subsequent polyacrylamide (PAA) gel purification and ethanol precipitation. RNA 5΄-ends were dephosphorylated using antarctic phosphatase (NEB) and labeled with [γ-32P]-ATP (Hartmann Analytic) using T4 polynucleotide kinase (NEB) according to manufacturer's protocols. Structural analysis of 10 000 cpm 5΄-end labeled RNA per reaction was performed via in-line probing (38 (link)) in the presence of 50 mM Tris-HCl (pH 8.5), 12 mM MgCl2, 0.1 mg/ml tRNA from E. coli MRE 600 (Roche Diagnostics) and 0–10 mM streptomycin. All purification steps were carried out on 12.5% or 15% denaturing PAA gels. Imaging of 32P-labeled RNA was performed with a Typhoon 9410 Variable Imager (GE Healthcare).
6
Radioactive RNA Labeling and Purification
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RNA was radioactively labelled by adding 80 μL of reaction mix containing 4 μL of 32P-γATP (10 μCi/μL; Hartmann Analytic), 2 μL of T4 Polynucleotide Kinase, 2 μL of RNasin and 20 μL of 50% PEG8000, in 1x R1 buffer. After 30 min at 37 °C, 1 μL of 100 mM ATP (ThermoFisher; cat. 10304340) was added, and the reaction left for an additional 15 min. A 5′ linker was then ligated by adding 4 μL of T4 RNA ligase 1 (NEB; cat. M0204L) and 4 μL of 100 μM barcoded linker. The reaction was performed for 3 h at 16 °C then 2 h at 25 °C. The 5′ linker was again produced by IDT. It has an inverted ddT at the 5′ end to increase stability and a barcode sequence at the 3′ end. Also at the 3′ end, there is a random 3-mer to allow collapsing of PCR duplicates. Following ligation, columns were washed three times with 0.5 mL of buffer WB1. RNA:protein complexes were then eluted by incubating beads with 50 μL of buffer BE (5 min at RT, shaking at 800 RPM), then spinning out eluate (1000 RCF for 1 min at RT). Elution was repeated, and eluates combined. RNA:protein complexes were then precipitated by adding 9 volumes of 100% ethanol and 3 μL of GlycoBlue co-precipitant (Invitrogen; cat. AM9516), and incubating samples overnight at −20 °C.
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