Author: Laneve, Pietro; Piacentini, Lucia; Casale, Assunta Maria; Capauto, Davide; Gioia, Ubaldo; Cappucci, Ugo; Di Carlo, Valerio; Bozzoni, Irene; Di Micco, Patrizio; Morea, Veronica; Di Franco, Carmela Antonia; Caffarelli, Elisa
Title: Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration Document date: 2017_1_31
ID: 1rw05x6m_34
Snippet: Analysis of cleavage product 3′-ends. Specific reaction products obtained by incubation of unlabeled P1 oligonucleotide with CG2145-or CG3303-expressing reticulocyte lysates were gel-purified after acrylamide fractionation, phenol-purified and redissolved in 30 mM Tris, pH 8.0, 15 mM MgCl 2 . The mixture was incubated for 45 min at 37 °C in the presence of 1.5 units/μ l of T4 polynucleotide kinase to remove the 2′ -3′ -cyclic phosphate 34.....
Document: Analysis of cleavage product 3′-ends. Specific reaction products obtained by incubation of unlabeled P1 oligonucleotide with CG2145-or CG3303-expressing reticulocyte lysates were gel-purified after acrylamide fractionation, phenol-purified and redissolved in 30 mM Tris, pH 8.0, 15 mM MgCl 2 . The mixture was incubated for 45 min at 37 °C in the presence of 1.5 units/μ l of T4 polynucleotide kinase to remove the 2′ -3′ -cyclic phosphate 34 . RNA was treated with kinase buffer or with alkaline phosphatase (Roche Applied Science) as negative controls. Extracted RNA was then labeled by T4 RNA ligase (New England Biolabs) for 5 h at 16 °C, in the presence of 5′ -[32 P] cytidine 3′ ,5′ -bisphosphate (Perkin Elmer Life Science) and analyzed on 20% polyacrylamide, 7 M urea gel.
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