RNA and DNA substrates were synthesized (IDT)-
RNA: 5′ GAAAUAUGGCGAGGAAAACUGAAAAAGGUGGAAAA 3′,
DNA_template: 5′ AF488:TTTTCCACCTTTTTCAGTTTTCCTCGCCATATTTC 3′,
DNA_non-template: 5′ GAAATATGGCGAGGAAAACTGAAAAAGGTGGAAAA 3′.
DNA:RNA hybrid and dsDNA were formed using 10 μM of each oligonucleotide, denatured at 95°C for 5 min, then cooled gradually to 21°C (Bio-Rad T100 Thermal Cycler). Reactions were assembled in 20 μL volume with purified protein and dsDNA or DNA:RNA hybrid in binding buffer containing 50 mM Tris pH 8.0, 100mM NaCl, 10 μg/ml BSA, 1 mM DTT, 0.1 mM EDTA, 5% Glycerol, and 2 μg yeast tRNA (Ambion cat# AM7119). Binding reactions were incubated at 30°C for 30 minutes and resolved on an 8% native polyacrylamide gel at 120V for 2 hours in 0.5 × TBE (44.5 mM Tris, 44.5 mM Boric Acid, 1 mM EDTA) at 4°C. Nucleic acids were visualized using the Amersham Typhoon Gel and Blot Imaging Systems (GE).
RNA: 5′ GAAAUAUGGCGAGGAAAACUGAAAAAGGUGGAAAA 3′,
DNA_template: 5′ AF488:TTTTCCACCTTTTTCAGTTTTCCTCGCCATATTTC 3′,
DNA_non-template: 5′ GAAATATGGCGAGGAAAACTGAAAAAGGTGGAAAA 3′.
DNA:RNA hybrid and dsDNA were formed using 10 μM of each oligonucleotide, denatured at 95°C for 5 min, then cooled gradually to 21°C (Bio-Rad T100 Thermal Cycler). Reactions were assembled in 20 μL volume with purified protein and dsDNA or DNA:RNA hybrid in binding buffer containing 50 mM Tris pH 8.0, 100mM NaCl, 10 μg/ml BSA, 1 mM DTT, 0.1 mM EDTA, 5% Glycerol, and 2 μg yeast tRNA (Ambion cat# AM7119). Binding reactions were incubated at 30°C for 30 minutes and resolved on an 8% native polyacrylamide gel at 120V for 2 hours in 0.5 × TBE (44.5 mM Tris, 44.5 mM Boric Acid, 1 mM EDTA) at 4°C. Nucleic acids were visualized using the Amersham Typhoon Gel and Blot Imaging Systems (GE).