Why does the activity of the enzyme vary from lot to lot?
Any topo preparation that passes our QC tests, has at least 2 units/ul based on decatenation assays with kDNA. Often we find that the activity is significantly greater (for example 4, 8 or 16 units/ul); however, since the enymes are unstable to dilution, we prefer to maintain concentrated stock enzyme and still sell it to the customer labeled conservatively. This is a good deal for the end user of course. On the other hand, we sometimes find that customers are confused by this since the activity seems to vary from lot to lot. One lot may be very very active (16 units/ul) and the next lot may be only 4 units/ul. If you need to standardize the assay from one lot the next, we suggest that you titer the activity in your own lab using your own assays. Another reason we sell the enzyme “hotter” than expected is that many researchers use them in cleavage assays which require higher levels of activity (compared to catalytic assays–see questions and discussion below).
What is the best way to store Topo Enzymes after we receive them?
Topo II is best kept at -70, while topo I and gyrase can be stored at -20 C. We suggest that you aliquot the enzyme into smaller fractions and try to minimize freeze thaw cycles. Both topo I and II may retain activity when stored in wet ice (0-4 C) for a few days, but not longer.
Different reagents in the kits are stored under differnt conditions. Why is this?
Some 10x buffers in particular are prone to be inactivated when subjected to freezing and thawing cycles. This is a serious problem with the topo II buffers containing ATP. In other cases, freeze/ thaw cycles of DNA substrates can cause their degradation. For example, kDNA and supercoiled DNA can be nicked or degraded by sequential freezing and thawing.
How should I proceed if the Topos purchased from TopoGEN appear to be inactive?
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.
For detecting Topo/DNA cleavages using drug screening kits, how much enzyme is required?
Usually these assays require 2 ul of undiluted enzyme; in other words more than 4 units of activity. In some cases you may be able to use 1 ul, but this depends upon the actual lot of enzyme being tested; as noted above, some lots are more active than others.
When should I use ethidium bromide gel separations?
Ethidium bromide (EB) is used to both visualize DNA bands by fluorescence and when included in the running buffer to resolve circular from nicked DNA forms. Clearly in both cases, EB (at 0.5ug/ml) is required for detection of bands. The only difference is when it is used (either during electrophoresis or after). It is essential that, in either case, you destain with distilled water for 30 minutes prior to photography. You should include EB in the gel and buffer whenever you want to resolve nicked circular DNA (Open circular or OC DNA) from covalently closed circular (CCC) DNA (Panel A). Note that CCC DNA can either be relaxed or supercoiled. Without EB, two possible patterns are possible. As shown in Panel B, OC DNA and relaxed CCC DNA are sometimes poorly resolved and may migrate close together (see Panel B on the right). Panel C show another possible pattern. In this case, topo relaxation generates a series of topoisomers (circular DNAs that differ only in linking number) that migrate as a set of bands between Supercoiled DNA and OC DNA. Depending on the conditions of gel electrophoresis (voltage or temperature and time) either pattern is possible. In cases where EB is included in the gel system, relaxed vs. supercoiled DNA separate very poorly whereas nicked OC DNA is cleanly resolved (see Panel A). Note that if you do not run the EB containing gel long enough, these two forms MAY NOT separate at all, in which case you cannot tell whether the enzyme was active or not (refer to Panel A). We recommend that you use EB gels (as in Panel A) whenever you are interested in detecting some sort of cleavage product from a plasmid DNA substrate. In contrast, use the non-EB gel system (panel B,C) whenever you wish to titrate relaxation activity since it is very easy to monitor loss of supercoiled CCC DNA.
1. Use EB in the gel during electrophoresis if you KNOW FOR SURE that the enzyme is active and your goal is to measure cleavage products.
2. Do not use the EB gel system if you are trying to titrate enzyme activity or if you are simply setting up the assay for the first time.
The kDNA prepartions we get from TopoGEN sometimes have what looks like a small bit of decantenated kDNA. Why is this?
Kinetoplast DNA (KDNA) is mitochondrial DNA from an insect tyrpanosome and is a conglomerate of mostly 2.5 kilobase minicircles interlocked as a collection of catenanes. Since the KDNA is isolated from the whole cell, there can be a carry over of host (genomic) DNA. The amount of host DNA will vary somewhat from lot to lot but should not exceed 20% of the total. A small amount of this high molecular weight cell DNA will not interfere with topo II assays because the decatenation products do not comigrate with the contaminating host DNA. In some cases, if the gels are run for very short times, under conditions where the resolution is poor, there may be complications; however, this can be corrected by running the dye front further down the gel. It is also important to include a KDNA marker (negative control) in each and every gel to be sure that you do not count stray bands as topo II activity.
Sometimes we find that one lot of kDNA may not suport Topo II activity as well as another lot. Why is ty is this?
KDNA must be purified from nuclear DNA using a density gradient of CsCl. One unfortunate characteristic of exposing KDNA catenanes to extremely high concentrations of CsCl is that contaminating heavy metals and undesirable ions can get trapped in the DNA ion atmosphere (estimated to be rich in counterions near the surface of the DNA helix). While this problem can result in very weak KDNA substrate activity with topo II, we screen each KDNA batch for this complication. We test 2 units of topo II with each new lot of KDNA; thus, the new lot must allow detection of 2 units at least. Some lots of kDNA that pass our screen may not be as robust as others and when trying to assay small amounts of topo II activity, any contamination problems may become more obvious. In these cases, please contact tecnnical support for assistance. What is the best way to resolve topoisomers in an agarose gel system? Run Chloroquine gels as follows: 1. Cast a 1-1.5% gel in 0.2ug/ml chloroquine in TPE buffer (36 mM Tris-Cl, pH7.8, 30mM NaH2PO4, 1 mM EDTA). 2. Run gel in TPE buffer at 15 volts for 15 hrs. at room temperature. 3. Stain with 0.5ug/ml Ehtidium bromide 4. Destain well (15-30 min) with H2O and photograph.
Why does the assay buffer supplied with Topo II & Gyrase sometimes go bad?
These buffers contain ATP which is somewhat unstable, particularly at the pH used in the 10x stock buffer. We cannot control this problem, however you should be aware that lack of strong topo II activity with positive controls (i.e. our enzymes), may be due to ATP degradation. In this case we recommend that you remake the buffer from the recipe given OR supplement with ATP.