There can be many reasons for low activity. Sometimes it is a bad tube of enzyme or a contaminant of some sort is masking the enzyme activity. In other cases, it is user error due to poor water quality, pipette errors or technical problems. Another problem can be related to degradation of buffers. As noted above, 10x buffers can inactivate with freeze thaw cycles. Sometimes, the enzyme is mishandled during shipping or after receipt. Concerning this point, it is always our goal is to keep your experiments running smoothly. Usually we can determine the most likely source of problems. In any event, the best course of action is to contact us by email with pictures of gels to allow us to determine the quality of your separation and general methods being used. For example, sometimes the gels are not run properly and the topoisomerase reaction products cannot be visualized effectively.
SPECIFIC RECOMMENDATIONS REGARDING ENZYME ACTIVITY PROBLEMS:
1. Contact us about the problem.
2. Reassay the enzyme testing 1 ul up to as much as 4 ul of enzyme in a 20 to 30 ul reaction.
3. Remake the 10x buffer and reassay the enzyme as above. Sometimes, buffers can lose potency and may not support enzyme activity.
4. When checking an enzyme that may have problems, try to keep it simple. Always be sure to run markers (linear, supercoiled plasmid or KDNA equivalents). Assay only a couple of enzyme concentrations (a low and high) but try not exceed 10% of the total reaction volume with stock enzyme (for example, no more that 4 ul in a 30 ul total reaction volume).
5. In terms of the gel separation method, if you are assaying relaxation of plasmid DNA, run your gel without ethidium bromide in the gel/buffer system. Stain with ethidium bromide AFTER you run the gel. This will make relaxation products much easier to visualize.
Why do I have to use more enzyme to assay for cleavage complexes with TopoGEN Drug Screening Kits (Topo I Drug Kit or Topo II Drug Kit)?Top
The enzymes are sold in catalytic units based upon a unit definition of relaxation (topo I) or decatenation (topo II). Both of these are based upon enzyme catalysis. Cleavage type assays measure a stoichiometric intermediate, the topo/DNA complex, which is related to catalytic activity but is not a turnover based assay. In other words, one is trapping a stoichiometric complex that actually uses up the enzyme. By definition, it will require more enzyme.