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

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.


  1. Kehinde on August 16, 2017 at 11:10 am


    This is urgent I mistakenly put -20C enzyme in -80C for about 3hrs, do I need to worry about its activity?

    • Dr Amanda Welch on August 23, 2017 at 3:12 pm

      Probably not. But I would thaw it slowly and run a test reaction with it first!

  2. bandhna on March 9, 2017 at 4:13 am

    i have to store enzyme glucose oxidase (already lyophiliized at -20 degree ) in laboratory
    can i store it in freezer ???
    please reply… its urgent help me …

  3. Hani on February 27, 2017 at 10:46 am

    We found our freezer alarming in the morning, and the tempreture was 0 degree instead of -20, we strore all of our restriction boxes and PCR kits there, do you think if they still working if they are at Zero degree over night?

  4. Jes on February 20, 2017 at 8:29 pm

    Hello !

    I forgot my DNase at room temperature for approximately 1 hour. Do you think that it is still usable ?

  5. HASAN on September 22, 2016 at 10:18 am

    Hi guys,
    I have just left my restriction enzymes on ice bucket for long approximately over night and I am really afraid and would like to ask you will they work perfectly again after I return them to the freezer?


    • Dr Amanda Welch on September 22, 2016 at 1:07 pm

      Hi Hasan,

      It really depends on your enzyme–I’ve had mixed success. To be entirely safe, you should discard the old enzymes and buy new. But I know that is really expensive.

      To test your enzymes, I would get about 1 microgram of your DNA, set up a digestion reaction in 50 microliters, and incubate it for an hour. Run the digested DNA out on an agarose gel with appropriate controls (ladder and uncut plasmid at the least). If your DNA is completely digested, then the enzyme is fine. If not, then toss it. Do this individually for all the enzymes you left out overnight.

      Good luck!

  6. Victoria on December 7, 2015 at 8:11 pm

    I’m curious how long people keep their restriction enzymes. Do they throw them out after the use by date? Check if they are still working before using something past their use by date?

    • Dr Amanda Welch on December 8, 2015 at 2:15 am

      I used enzymes past their use by date all the time. I wouldn’t usually check them before using them in an experiment (unless the sample was precious or something like that).

  7. riya on October 20, 2015 at 6:56 am

    should we always keep th enzyme vial (powder form and stored at -20 degree celsius) at room temperature to acclamatize before weighing?

  8. Pavel Zach on May 8, 2013 at 6:08 pm

    Another opinion (focused on restriction enzymes):

    • user-96364 on May 9, 2013 at 1:34 pm

      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!

  9. Jarle Pahr on May 8, 2013 at 4:28 pm

    It would be good with some references.

    • 5 to 16 chars on May 10, 2013 at 12:42 am

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

      • Nick Oswald on May 10, 2013 at 11:42 am

        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!

        • hafeez dawar on May 11, 2017 at 9:54 am

          sir,please tell me that if we are keeping enzymes constantly at -20 degreecentigrade than would there iz no chance for the enzymes to become denature at that temperature

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