A total of 10 μl DNA cleavage reaction consists of 20 nM dsDNA substrates, 200 nM TnpB, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and the reaction was initiated by incubation at 60 °C, 70 °C or 75 °C for indicated periods. Then the reaction was quenched by adding 2 μl 6x SDS DNA loading dye (1% SDS, 100 mM EDTA, 60% glycerol, and 0.03% bromophenol blue) and reaction products were analyzed by 1% agarose gel electrophoresis.
To identify the cleavage site of TnpB7 on the dsDNA target flanked by the TTTAA TAM, we incubated 200 nM TnpB with 2 μg linear dsDNA target in the reaction buffer as described above. The reaction was incubated at 70 °C for 1 h. Reaction products were resolved by TBE-agarose gel electrophoresis. Two cleavage products were recovered from the agarose gel using the gel extraction kit (Omega) and sent for run-off DNA sequencing. The cleavage pattern of TnpB was determined by aligning the sequencing results with the target sequence.
The reactions used for assessing trans-cleavage activities of TnpB consist of 50 nM 5’ FAM-labeled ssDNA substrates (35 nt) or dsDNA substrate (62 bp), 300 nM TnpB7 and 50 nM activator DNA (ssDNA or dsDNA target DNA) of TnpB7. The reactions were incubated at 75 °C for 10 min. The resulting products were resolved by 15% urea polyacrylamide gel electrophoresis and visualized with an Amersham ImageQuant 800 biomolecular imager (Cytiva).
To identify the cleavage site of TnpB7 on the dsDNA target flanked by the TTTAA TAM, we incubated 200 nM TnpB with 2 μg linear dsDNA target in the reaction buffer as described above. The reaction was incubated at 70 °C for 1 h. Reaction products were resolved by TBE-agarose gel electrophoresis. Two cleavage products were recovered from the agarose gel using the gel extraction kit (Omega) and sent for run-off DNA sequencing. The cleavage pattern of TnpB was determined by aligning the sequencing results with the target sequence.
The reactions used for assessing trans-cleavage activities of TnpB consist of 50 nM 5’ FAM-labeled ssDNA substrates (35 nt) or dsDNA substrate (62 bp), 300 nM TnpB7 and 50 nM activator DNA (ssDNA or dsDNA target DNA) of TnpB7. The reactions were incubated at 75 °C for 10 min. The resulting products were resolved by 15% urea polyacrylamide gel electrophoresis and visualized with an Amersham ImageQuant 800 biomolecular imager (Cytiva).