The ethanol/gradient mixture of input and gradient material was incubated overnight at −20°C and pelleted in a benchtop centrifuge for 20 min at 16 000 x g and 4°C. The pellets were TRIzol (Invitrogen) extracted following the manufacturer's protocols. The resulting RNA was either sequenced by the Dresden Genome Center on an HiSeq2000 (Illumina) platform or reverse transcribed using random hexamer primers and RevertAid Premium reverse transcriptase (Fermentas), according to the manufacturer's protocols. Quantitative PCR (qPCR) was conducted on a Mx3000P qPCR system (Stratagene) using the ABsolute QPCR SYBR Green mix (Thermo) and gene-specific primers (sequences available upon request). All gradient data were normalized to the FFluc spike-in mRNA control. For bulk poly(A) tail measurements, total RNA was isolated from hand-picked adults using the TRIzol method. One microgram of total RNA was used in the 3′ end labeling assay and processed according to the published methods (26 (link)), with the only exception that un-incorporated α32P-Cordycepin (Perkin Elmer) was removed using Mini Quick Spin Columns (Roche).
α32p cordycepin
Manufactured by PerkinElmer
Sourced in United States
α32P-Cordycepin is a radioactive compound used in various laboratory applications. It is a nucleoside analog that incorporates the radioactive isotope of phosphorus, 32P, into its structure. This product is commonly utilized as a tracer or label in techniques such as Northern blotting, RNA labeling, and DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
γ 32p atp, by PerkinElmer (4 mentions)
Terminal transferase, by New England Biolabs (2 mentions)
T4 pnk, by Thermo Fisher Scientific (2 mentions)
5 irdye 700, by Integrated DNA Technologies (2 mentions)
5 irdye 800 labelled, by Integrated DNA Technologies (2 mentions)
3 6fam labelled oligonucleotides, by Merck Group (2 mentions)
Mini quick spin column, by Roche (1 mentions)
Revertaid premium reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Absolute qpcr sybr green mix, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Unlabeled nucleotides, by GE Healthcare (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
α 32p dctp, by PerkinElmer (1 mentions)
5 protocols using α32p cordycepin
1
RNA Extraction and Sequencing Protocol
2
Radiolabeling of DNA Substrates
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Unlabeled nucleotides were purchased from GE Healthcare. Labeled nucleotides [α32P]-Cordycepin (3′-dATP; 3000 Ci/mmol) and [γ32P]-ATP (3000 Ci/mmol) were obtained from Perkin Elmer Life Sciences. Substrates were radiolabeled at the 3′ end with [α32P]-Cordycepin and terminal deoxynucleotidyl transferase (TdT) or at the 5′ end with [γ32P]-ATP and T4 polynucleotide kinase (T4PNK), as indicated. TdT, T4PNK, Escherichia coli uracil DNA Glycosylase (UDG), E. coli EndoIII (EndoIII), human AP endonuclease I (hAPE1) and Ribonuclease HII (RNaseHII), were from New England Biolabs. Thrombin was obtained from Novagen. BsuKu and BsuLigD were purified as described (25 (link)). The N-terminal Ligase domain (LigDom, residues 1–320) of BsuLigD was purified as described (26 (link)).
3
Synthesis and Labeling of DNA Substrates
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Synthetic substrates were prepared essentially as described (82 (link)), employing PAGE-purified ssDNA oligonucleotides. All the oligonucleotides are listed in Supplementary Table S3 and the strand composition of each substrate is described in Supplementary Figure S1 . Briefly, labelled and unlabelled oligonucleotides were mixed in a 1:3 ratio, boiled in a water bath, cooled down to room temperature overnight and fully ligated substrates were purified from 10% polyacrylamide gels. For radioactive substrates, oligos were either 5′-end-labelled with [γ-32P]-ATP (3000 Ci/mmol, Perkin Elmer) and T4 PNK (Thermo Fisher) or 3′-end-labelled with [α-32P]-cordycepin (3000 Ci/mmol, Perkin Elmer) and terminal transferase (New England Biolabs). For fluorescent substrates, 5′-IRDye 700, 5′-IRDye 800-labelled (Integrated DNA Technologies), 5′-6FAM or 3′-6FAM-labelled oligonucleotides (Sigma-Aldrich) were employed. For biotinylated substrates, 5′-biot-X01 oligonucleotide (Sigma-Aldrich) was employed. Unlabelled substrates were prepared with equimolecular mixtures of oligonucleotides and purified from native 10% polyacrylamide gels by UV-shadowing. When appropriate, oligonucleotides containing one or three consecutive phosphorothioate (SP) linkages (Sigma-Aldrich) were employed.
4
Purification and Characterization of DNA Repair Enzymes
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Oligonucleotides were from Oligos Etc, Inc. (Wilsonville, OR, USA) and The Midland Certified Reagent Co. (Midland, TX, USA), Inc. [α-32P]dCTP and [α-32P]Cordycepin (3000 Ci/mmol), a substitute of ddATP, and [γ-32P]ATP (6000 Ci/mmol) were from PerkinElmer (Waltham, MS). Optikinase and terminal deoxynucleotidyl transferase were from USB Corp. (Cleveland, OH, USA) and Fermentas Inc. (Hanover, MD, USA), respectively. Protease inhibitor complete (EDTA-free) was from Roche Molecular Diagnostics (Pleasanton, CA, USA). Leupeptin, aprotinin, and phenylmethylsulfonyl fluoride were from Calbiochem (La Jolla, CA, USA). Recombinant human DNA pol β was overexpressed and purified as described previously (46 (link)). Human recombinant APE1, uracil-DNA glycosylase (UDG) with 84 amino acids deleted from the amino-terminus and DNA ligase I were purified as described previously (47 (link)–49 (link)).
5
Preparation of Synthetic DNA Substrates
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Synthetic substrates were prepared essentially as described (80) , employing PAGE-purified ssDNA oligonucleotides. All the oligonucleotides are listed in Supplementary Table S3 and the strand composition of each substrate is described in Supplementary Table S4. Briefly, labelled and unlabelled oligonucleotides were mixed in a 1:3 ratio, boiled in a water bath, cooled down to room temperature overnight and fully-ligated substrates purified from 10% polyacrylamide gels. For radioactive substrates, oligos were either 5'-end labelled with [γ-32 P]-ATP (3000 Ci/mmol, Perkin Elmer) and T4 PNK (Thermo Fisher) or 3'-end labelled with [α-
32 P]-cordycepin (3000 Ci/mmol, Perkin Elmer) and terminal transferase (New England Biolabs). For fluorescent substrates, 5'-IRDye 700, 5'-IRDye 800-labelled (Integrated DNA Technologies) or 3'-6FAM-labelled oligonucleotides (Sigma-Aldrich) were employed.
Unlabelled substrates were prepared with equimolecular mixtures of oligonucleotides and purified from native 10% polyacrylamide gels by UV-shadowing. When appropriate, oligonucleotides containing 1 or 3 consecutive phosphorothioate (SP) linkages (Sigma-Aldrich) were employed.
32 P]-cordycepin (3000 Ci/mmol, Perkin Elmer) and terminal transferase (New England Biolabs). For fluorescent substrates, 5'-IRDye 700, 5'-IRDye 800-labelled (Integrated DNA Technologies) or 3'-6FAM-labelled oligonucleotides (Sigma-Aldrich) were employed.
Unlabelled substrates were prepared with equimolecular mixtures of oligonucleotides and purified from native 10% polyacrylamide gels by UV-shadowing. When appropriate, oligonucleotides containing 1 or 3 consecutive phosphorothioate (SP) linkages (Sigma-Aldrich) were employed.
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