Oligomer End-Labeling and Annealing Protocol

T4 polynucleotide kinase (NEB) was used to end label oligomers using γ-³²P ATP in T4 PNK buffer (NEB) as described before^{38 (link)}. Reaction was incubated for 1 h at 37 °C and then stopped using 10 mM Tris-1 mM EDTA buffer. Labelled substrates were purified using Sephadex G-25 column and then stored at −20 °C until use. Annealing of complementary oligomers was done by boiling oligomers for 10 minutes in the presence of 100 mM NaCl and 1 mM EDTA, in 1:5 ratio (labelled: unlabelled)^{37 (link),38 (link)}.

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Nilavar N.M., Paranjape A.M, & Raghavan S.C. (2020). Biochemical activity of RAGs is impeded by Dolutegravir, an HIV integrase inhibitor. Cell Death Discovery, 6, 50.

Publication 2020

T4 polynucleotide kinase T4 PNK buffer

Buffer Done Edta Nacl Polynucleotide kinase Sephadex g 25 Tris buffer

Corresponding Organization :

Other organizations : Indian Institute of Science Bangalore

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Variable analysis

independent variables

T4 polynucleotide kinase (NEB) used to end label oligomers
γ-32P ATP used for labeling
T4 PNK buffer (NEB) used for the reaction
Reaction incubation time of 1 h at 37 °C
Annealing of complementary oligomers by boiling in the presence of 100 mM NaCl and 1 mM EDTA, in 1:5 ratio (labelled: unlabelled)

dependent variables

Labelled substrates after purification using Sephadex G-25 column

control variables

Tris-1 mM EDTA buffer used to stop the reaction
Labelled and unlabelled oligomers used in 1:5 ratio for annealing

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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