Using Enzymes at the Bench — Keep it in the cooler? On ice? or at RT?

by on 8th of May, 2013 in Cloning & Expression
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I am a research coordinator/scientific writer and editor currently based in Perth, Australia.
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Have you ever needed an enzyme but not had an ice bucket?  Have you been tempted to just grab the enzyme out of the freezer, take what you need and quickly return the tube to the freezer?  Do enzymes really need to be kept on ice all of the time?  I would say, Yes!

Enzymes used in a research laboratory range from restriction enzymes used for plasmid digests, reverse transcriptases for cDNA synthesis reactions to polymerases used for PCR. Like many laboratory reagents enzymes are expensive! It is good laboratory housekeeping to maintain their longevity through proper storage and use. This doesn’t just make good economics, it also ensures the quality of your experiments.  And it will keep you in the good graces of your labmates and PI!

Enzymes need to be stored and used at their optimum temperatures. Enzymes are generally stored in glycerol at -20C. This prevents them from freezing completely, which causes protein denaturation and results in a loss of activity. Enzymes will also denature if they are warmed up, repeatedly exposed to temperature fluctuations, or stored at room temperature for extended periods of time. Therefore, enzymes need to be kept cold at all times, even if you are just pipetting out of them quickly.

A little organization will help you set up your reactions without exposing enzymes to warmer temperatures.  Make sure you have everything you need on hand prior to starting.  If you are running an experiment that requires a programmed machine, make sure you book the machine and give yourself plenty of time to get everything set up.  Program the machine prior to setting up your reactions. It is good practice to set up your reaction(s) by adding all other components first then adding your enzyme last. This is particularly important when working with Taq for PCR reactions; the polymerase can start working immediately so it is important to add the enzyme and get your samples into the machine quickly. Keep your enzymes in the freezer until just before addition. Bring them over to your bench in an ice bucket or enzyme cooler and return them to the freezer immediately after using.

So, to keep your enzymes happy, store them in the freezer and keep them on ice when working on the bench.

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5 thoughts on “Using Enzymes at the Bench — Keep it in the cooler? On ice? or at RT?”

  1. Avatar of Jarle Pahr Jarle Pahr says:

    It would be good with some references.

    1. I agree!!! We are scientists, aren't we? Can we not have evidence-based research, rather than assertions?

      1. Avatar of Nick Oswald Nick Oswald says:

        Thanks for the comments guys. This is a balance for us, as some of the things we write about are not in the literature and are just knowledge and opinions acquired from experience in the lab.

        But in this case is there is some literature evidence out there, so we will get something written up on this topic, with some references. Thanks!

    1. Avatar of user-96364 user-96364 says:

      Haha- you beat me to it Pavel. March JB is my current boss by the way.

      I think it's tempting to become paranoid about enzymes – often the data concerning how sensitive they are is incomplete. Personally – I do use a nice convenient cool block for any important enzymes (if they are generally -20 stored). To be honest, I don't really care if they are stable or not – I'd just abide by what the company who makes them says!

      For standard PCR, I don't think it's worth getting too fussy about 'rushing to the thermal cycler'. I've made the mistake of forgetting to press 'go' on the PCR machine (in my early days) and my reactions were left at room temp for several hours – everything worked 'as well as was needed' once I did press go, so no big shakes. So relax, everything will probably be fine!

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