Tnt t7 sp6 kit

Recombinant proteins were synthesized using a coupled transcription-translation system (Promega TnT T7/SP6 kit) and incubated with 0.5 ng radiolabelled oligonucleotide probe in binding buffer (20mM Tris pH 7.6, 75mM KCl, 0.25mg/ml, 1mM DTT, 10% glycerol) at room temperature for 10 minutes. For competition experiments, excess of unlabelled oligonucleotide probes was added and incubated for 10 minutes prior to addition of radiolabelled oligonucleotide probes. Binding reactions were analysed on 6% 0.5X TBE polyacrylamide gels electrophoresed at 150V; gels were dried prior to detection of signal by autoradiography. Probes used were, M10-c: GATGACTGCCCTTATTTGACC and M10mut: GATGACTGggaggATTTGACC.

Recombinant proteins were synthesized using a coupled transcription-translation system (Promega TnT T7/SP6 kit) and incubated with 0.5 ng radiolabelled oligonucleotide probe in binding buffer (20 mM Tris [pH 7.6], 75 mM KCl, 0.25 mg/ml, 1 mM DTT, and 10% glycerol) at room temperature for 10 min. For competition experiments, the excess of unlabelled oligonucleotide probes was added and incubated for 10 min prior to the addition of radiolabelled oligonucleotide probes. Binding reactions were analyzed on 6% 0.5× Tris-borate-EDTA (TBE) polyacrylamide gels electrophoresed at 150 V; gels were dried prior to detection of signal by autoradiography.