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γ 32p atp 6000 ci mmol

Manufactured by PerkinElmer

[γ-32P]-ATP (6000 Ci/mmol) is a radioactively labeled nucleotide used for various biochemical and molecular biology applications. It serves as a tracer and labeling agent for studying enzymatic reactions, nucleic acid synthesis, and protein-nucleic acid interactions.

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6 protocols using γ 32p atp 6000 ci mmol

1

Radioisotope-Labeled DNA Repair Assay

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Reagents were purchased from the following suppliers and were of the highest purity available. Oligonucleotides were purchased from Integrated DNA Technologies (IDT, Coralville, IA) or, in the case of DHT-containing Oligonucleotides, from Midland Certified Reagent Co. (Midland, TX). Uracil DNA glycosylase (UDG), and T4 DNA polynucleotide kinase (T4 PNK), formamidopyrimidine DNA glycosylase (Fpg) and Endonuclease III (Nth) were from New England Biolabs (Ipswich, MA). [γ-32P]-ATP (6000 Ci/mmol) was purchased from PerkinElmer. C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA), and BS Poly-prep columns were obtained from BioRad (Hercules, CA). Acrylamide/bis-acrylamide 19:1 (40% Solution/Electrophoresis) was purchased from Fisher Scientific (Waltham, MA), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Quantification of radioactivity in polyacrylamide gels was carried out using a Personal Molecular Imager (BIORAD) with Quantity One software (v.4.6.5).
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2

Reconstitution of Lipid Bilayers

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All chemicals including KCl, Tris-HCl, MgCl2, pentane, sephadex, and hexadecane were obtained from Sigma-Aldrich (St. Louis, MO, USA) and were used as received. The compound 1,2-diphytanoyl-sn-glycero-3-phosphocholine used for lipid bilayer formation was from Avanti Polar Lipids (Alabaster, AL, USA) and was used without further purification. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). All enzymes were purchased from New England Biolabs (Ipswich, MA, USA). [γ-32P]-ATP (6000 Ci/mmol) was purchased from Perkin Elmer. Quantification of radioactivity in polyacrylamide gels was carried out using a Personal Molecular Imager (BIORAD) with Quantity One software (v.4.6.5).
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3

Oligonucleotide Synthesis and Labeling

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Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Uracil DNA glycosylase (UDG), ϕ29 DNA polymerase, and T7 DNA polymerase were from New England Biolabs (Ipswich, MA). A mixture of the four 2′-deoxynucleotide triphosphates (dNTP) was purchased from Promega (Madison, WI). [γ-32P]-ATP (6000 Ci/mmol) was purchased from Perkin-Elmer. C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA), and BS Poly-prep columns were obtained from BioRad (Hercules, CA). Acrylamide/bis-acrylamide 19:1 (40% Solution/Electrophoresis) was purchased from Fisher Scientific (Waltham, MA), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
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4

Preparation of Radiolabeled RNA Substrates

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RNAs for dicing assays (Table S2, Integrated DNA Technologies) were 5′ 32P radiolabeled using [γ−32P]ATP (6000 Ci/mmol) (PerkinElmer, Waltham, MA) and T4 polynucleotide kinase (NEB, Ipswich, MA), and then gel-purified. Substrates containing two 32P-radiolabeled phosphates were prepared by DNA-splinted ligation (Moore and Sharp, 1993 (link); Moore and Query, 2000 (link)). Synthetic hairpins for substrate competition assays (Table S2) were ordered from Integrated DNA Technologies as HPLC-purified RNAs.
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5

Synthesis and Characterization of Radiolabeled Oligonucleotides

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Oligonucleotides were purchased from Integrated DNA Technologies. All enzymes were purchased from New England Biolabs (Ipswich, MA, USA). [γ-32P]-ATP (6000 Ci/mmol) was purchased from Perkin Elmer. C-18 sep-pak cartridges were from Waters. BS Polyprep columns were purchased from BioRad. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) hydrochloride was from Tocris Bioscience (Ellisville, MO, USA). Nuclease P1 and phosphodiesterases 1 and 2 were from Sigma-Aldrich (St. Louis, MO, USA). Alkaline phosphatase and proteinase K were from New England Biolabs (Ipswich, MA, USA). Quantification of radioactivity in polyacrylamide gels was carried out using a Personal Molecular Imager (BioRad) with Quantity One software (v.4.6.5). All other reagents were purchased from Sigma-Aldrich. DMBAA (dimethylbutylammonium acetate) solutions used in the ESI-MS experiments was prepared as follows: a stock of N,N-dimethylbutyl amine (7.125 M) was diluted to 100 mM with water and adjusted to pH 7.1 with glacial acetic acid.
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6

Radioactive Oligonucleotide Purification and Analysis

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Oligonucleotides were purchased from Integrated
DNA Technologies (Coralville, IA). Hydralazine hydrochloride, 2-deoxy-d-ribose, sodium hydroxide, and other chemicals were purchased
from Sigma-Aldrich (St. Louis, Mo) and used without further purification.
The enzyme uracil DNA glycosylase (UDG) was purchased from New England
Biolabs (Ipswich, MA). [γ-32P]-ATP (6000 Ci/mmol)
was purchased from PerkinElmer (Waltham, MA). C18 Sep-Pak cartridges
were purchased from Waters (Milford, MA), and BS Polyprep columns
were obtained from BioRad (Hercules, CA). Measurement of radioactivity
in polyacrylamide gels was carried out using a Personal Molecular
Imager (Bio-Rad) with Quantity One software (v.4.6.5).
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