Biotinylated RNA fragments were generated using in vitro transcription MegaScript T7 (ThermoFisher Scientific cat # AM1334) and biotinylated-14CTP (ThermoFisher Scientific cat # 19519016) as per manufacturer’s instructions. Fragment sizes and purity were confirmed by Northern blot. RNAs were incubated with nuclear lysates produced as outlined in Friend et al. (2007) (link). Incubations shown are for 30 minutes. The following buffer was used: 1.5 mM MgCl2, 0.25 mM ATP, 1 mM cordycepin, 20 mM phosphcreatine, 2 units of RNase out. The reaction was stopped with G50 (20 mM TRIS, pH 7.5, 2 mM EDTA 200 mM NH4OAc, 0.25% SDS and RNase out). This was followed by proteinase K treatment for 30 min at 55C (ThermoFisher Scientific cat # AM2548). RNA was extracted using phenol / chloroform (ThermoFisher Scientific cat # 15596026) and Northern blots were carried out as described in Culjkovic et al. (2005) (link); Friend et al. (2007) (link), and Topisirovic et al. (2002) (link).
Biotinylated 14ctp
Manufactured by Thermo Fisher Scientific
Biotinylated-14CTP is a labeled nucleotide analog used in various molecular biology applications. It contains a biotin moiety and a radioactive carbon-14 label. This product can be used for labeling and detecting nucleic acids during experiments, such as Northern blotting, dot blotting, and in vitro transcription.
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2 protocols using biotinylated 14ctp
1
Biotinylated RNA Fragments Binding Assay
2
Biotinylated RNA Fragments Binding Assay
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Biotinylated RNA fragments were generated using in vitro transcription MegaScript T7 (ThermoFisher Scientific cat # AM1334) and biotinylated-14CTP (ThermoFisher Scientific cat # 19519016) as per manufacturer’s instructions. Fragment sizes and purity were confirmed by Northern blot. RNAs were incubated with nuclear lysates produced as outlined in Friend et al. (2007) (link). Incubations shown are for 30 minutes. The following buffer was used: 1.5 mM MgCl2, 0.25 mM ATP, 1 mM cordycepin, 20 mM phosphcreatine, 2 units of RNase out. The reaction was stopped with G50 (20 mM TRIS, pH 7.5, 2 mM EDTA 200 mM NH4OAc, 0.25% SDS and RNase out). This was followed by proteinase K treatment for 30 min at 55C (ThermoFisher Scientific cat # AM2548). RNA was extracted using phenol / chloroform (ThermoFisher Scientific cat # 15596026) and Northern blots were carried out as described in Culjkovic et al. (2005) (link); Friend et al. (2007) (link), and Topisirovic et al. (2002) (link).
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