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)).
Protocol detail
Radiolabeling of DNA Substrates
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Cordycepin
E coli
Endonuclease i
Human
Ligase
Nucleotides
Polynucleotide kinase
Ribonuclease hii
Terminal deoxynucleotidyl transferase
Thrombin
Uracil dna glycosylase
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Variable analysis
independent variables
- Radiolabeled substrates at the 3' end with [α^32P]-Cordycepin and terminal deoxynucleotidyl transferase (TdT)
- Radiolabeled substrates at the 5' end with [γ^32P]-ATP and T4 polynucleotide kinase (T4PNK)
- Outcomes or measurements related to the use of the radiolabeled substrates and enzymes
- Unlabeled nucleotides purchased from GE Healthcare
- Radiolabeled nucleotides [α^32P]-Cordycepin (3'-dATP; 3000 Ci/mmol) and [γ^32P]-ATP (3000 Ci/mmol) obtained from Perkin Elmer Life Sciences
- Enzymes TdT, T4PNK, Escherichia coli uracil DNA Glycosylase (UDG), E. coli EndoIII (EndoIII), human AP endonuclease I (hAPE1) and Ribonuclease HII (RNaseHII) from New England Biolabs
- Thrombin obtained from Novagen
- BsuKu and BsuLigD purified as described (25)
- N-terminal Ligase domain (LigDom, residues 1–320) of BsuLigD purified as described (26)
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