Topoisomerase Enzyme | 고마바이오텍

Relaxation assays were carried out in a final volume of 25 µl in recombinant topo I reaction buffer (10X reaction buffer, supplied with this product is: 100 mM Tris-Cl, pH 7.9, 1.5 M NaCl, 1% BSA, 1 mM Spermidine, 50% glycerol).   Supercoiled plasmid DNA was included at 0.25 µg/reaction.  Reactions terminated with 5 µl (per 20 µl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products were analyzed on a 1% native agarose gel.  Under these conditions, relaxation activity is easily detectable (typically we see 10-40 units of activity per ul).  

Relaxation assays were carried out in a final volume of 25 μl in topo I reaction buffer (10X reaction buffer, supplied with this product is: 100 mM Tris-Cl, pH 7.9, 1.5 M NaCl, 1% BSA, 1 mM Spermidine, 50% glycerol).  Supercoiled plasmid DNA was included at 0.25 μg/reaction.  Reactions terminated with 5 μl (per 20 μl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol).  Reaction products were analyzed on a 1% native agarose gel.  Under these conditions, relaxation activity was not detectable with up to 0.5 ug of mutant protein.

Decatenation assays are carried out using kinetoplast DNA substrate in a final volume of 20-30 µl in topo II reaction buffer (50 mM Tris-Cl, pH 8.0, 120 mM KCl, 10 mM MgCl2. 0.5 mM ATP, 0.5 mM dithiothreitol) with 0.2 µg KDNA.  Reactions are terminated with 5 µl (per 20 µl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products are analyzed on a 1% agarose gel containing 0.5 ug/ml ethidium bromide.  Resolution of a 2.5 kb DNA minicircle decatenated product can be easily monitored with a hand held UV light source while the gel is running.

Reactions contained the following (20 ul): 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 10 mM DDT, 100 mM potassium glutamate, 40 uM ATP, 50 ug BSA/ml, 0.2 ug kDNA.  Reactions were terminated after 30 min at 37°C with SDS (1%) followed by proteinase K digestion (50 ug/ml, 15 min at 56°C).  Reaction products were run on a 1% agarose gel containing 0.5 ug/ml ethidium bromide (monitored with hand held UV light to resolve decatenation products), destained with distilled water (20 min room temperature) and photographed.