Probably this question seems fool to you, but I am quite new in the research area and there are a lot of experiments I have never done. My question regards the amount of buffer, enzyme, incubation time and incubation temperature that are most suitable for a particular experiment. I know that the manufacturer (that produces a specific enzyme) releases all the indication regarding these aspects. Indeed, during my master's I have always followed the manufacturer indication to set an endonuclease reaction. However, in the lab where I actually am I have been prompted to reason that not always following the manufacturer indications yields the desired results and that the best practice would be performing an enzyme assay to test the appropriate amount of enzyme to use, temperature, incubation time and so on. Now, the question is: how can I do it? Simply preparing several endonuclease reaction mixtures in which I use different parameters, but maintaining unchanged the amount of DNA? And how much DNA would you suggest to use to not waste a lot of it?
Should I use the DNA that I want to digest for my experiment or there is a DNA sample that is mostly used for the enzymatic assay. I would really appreciate it if you could reply to all my questions and maybe tell me how you set your own enzyme assay experiments.
In general terms, not just for endonucleases, there are a variety of adjustable parameters in an enzyme reaction, some of which you mentioned. Optimizing the conditions can be time-consuming and, if you have to purchase expensive reagents, also expensive. If it is not necessary because the manufacturer has provided suitable conditions, I would not undertake it.
However, if you are determined to do it, here are some of the conditions you have to consider:
Enzyme concentration
Substrate DNA concentration
Cofactor concentration (such as Mg2+), if needed
Buffer type, pH and concentration
Salt type and concentration
Temperature
Reaction time
In the case of a restriction digestion, the goal is to run the reaction to completion, so that is the endpoint you are aiming for. The assay is going to be agarose gel electrophoreses, which is a relatively low-throughput method, so you have to work out how to obtain the desired information with the minimal number of experiments. This is where a fractional factorial design strategy is useful. You may be also able to settle on some of the parameters based on existing knowledge of the enzyme, rather than starting from scratch. For example, you may know that the enzyme does not require a cofactor and that it works best at 37oC, so you don't have to vary those parameters.
Enzyme concentration, substrate concentration and reaction time are tied together, of course. You may be able to choose a maximal enzyme concentration based on the cost of the enzyme and the desire to limit the glycerol concentration coming from the stock solution (which slows down the reaction).
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