A total of 50 ng ChIPed DNA was denatured incubating for 10 min at 99°C with 0.4 N NaOH, and 10 mM EDTA. Denatured DNA samples were applied to hybond N+ (Amersham; RPN119B) membrane using a dot-blot apparatus connected to a vacuum source and washed with 2× SSC once. DNA was denatured on a membrane using 1.5 M NaCl/0.5 N NaOH for 10 min and then neutralized using 1.5 N NaCl/0.5 M Tris pH 7. DNA was UV crosslinked using a UV Stratagene crosslinker. Membrane was prehybridized by rapid hybridization buffer (Amersham; RPN1635) for 1 h and incubated over night with radiolabeled telomere (TTAGGG)3 or Alu probe (5′-CGGGAAGCAGAGGTTGTAGTGAGCC). Reaction mixture was 1 μl telomere restriction fragment, 13.5 μl H2O, 2 μl T4 PNK buffer, 2.5 μl 32P-γATP and 1 μl T4-polynucleotide kinase (New England Biolabs) at 37°C. The membrane was washed the next day once with 0.1% SDS/2× SSC buffer and twice with 0.1% SDS/1× SSC. Radiolabeled membranes were exposed to a phosphorimager (Fuji), and signal intensities were quantified after 3 days exposure.
32p γatp
Manufactured by New England Biolabs
32P-γATP is a radioactive nucleotide used in various molecular biology and biochemical applications. It is a gamma-labeled form of adenosine-5'-triphosphate (ATP) where the gamma phosphate group is labeled with the radioactive isotope phosphorus-32 (32P). This product can be used as a substrate or label for enzymatic reactions, nucleic acid labeling, and other analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rpn119b, by Cytiva (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
T4 pnk buffer, by New England Biolabs (1 mentions)
Phosphorimager, by Fujifilm (1 mentions)
Bluejuice gel loading buffer, by Thermo Fisher Scientific (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
2 protocols using 32p γatp
1
Telomere Length Quantification by Dot-Blot Hybridization
2
Pre-miRNA Binding Assay with NF90/45
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Pre-miRNAs (1 pmol for each) were prepared as above from microprocessing assays and treated using CIP (NEB) in a 20 μl reaction system and then inactivated at 80°C for 2 min. The pre-miRNAs were then radioactively labeled as follows: 20 μl of de-phosphorylated RNA, 3 μl of 10x T4 PNK buffer, 3 μl of 32P-γ-ATP, 1 μl of T4 PNK (NEB) in a 30 μl reaction system and incubated at 37°C for 30 min. RNA were then purified by G-25 spin column (Fisher Scientific) and EDTA was added to 0.1 mM. RNA was heated to 95°C for 2 min and immediately chilled on ice. These oligos were incubated with different amounts (0, 0.5 or 2 ug) of recombinant NF90/45 proteins, 4 μl of 5× EMSA binding buffer (5×: 100 mM HEPES pH 7.9, 375 mM KCl, 2.5 mM DTT, 0.05% Tween 20, 50% glycerol), and water up to 20 μl. The binding reaction was incubated on ice for 30 min, followed by the addition of 2 μl of BlueJuice gel loading buffer (Invitrogen). Samples were then resolved on the pre-run 6% TBE retardation gel (ThermoFisher) at 100 V and then imaged.
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