5 replies to this topic

[Biog]

Hi,

I am wondering about the "strict caution" about enzyme storing at -20°C. Manufacturers often say that we have to keep enzymes (e.g Taq) upon reception at -20°C. Yet, this enzyme is used in PCR at 95°C for at least 2 h and stay active after alternative cycles of 95/55°C! This means that its stability is very high.
Also, if we look at dishwashers liquid, all enzymes are kept at room temperature and stay active!
So, my question, is my boss right to panic when I forget the Taq on bench for some minutes?

Edited by Biog, 21 November 2011 - 05:23 PM.

[bob1]

Taq and many other enzymes do degrade quite quickly at room temperature, a few minutes are probably OK, but certainly hours are not.  At 95 deg taq degrades quite quickly, I think that it is only stable for a couple of minutes.  The enzymes in dishwashers etc are all stabilised or are forms that are resistant to degradation.  If you want to look into stable enzymes - RNase is a good place to start.

So as not to leave it at room temperature, it is good practice to keep it on ice at all times.

[Biog]

Thanks for reply.
You say that Taq is stable for a couple of minutes, how is that in a PCR run that lasts ~ 1h30? If it is stable only for a couple of minutes, this means that DNA will not be amplified efficiently!
During PCR run, Taq is active to amplify DNA; if it is degraded, there will be no DNA amplified, or only very small quantity!

[hobglobin]

Taq from Invitrogen has this half-life times:

5 minutes at 97.5°C
40 minutes at 95°C
120 minutes at 92.5°C

I guess Taqs from other companies have similar values. Therefore higher temperatures are not that problem for a short time. But anyway this are minutes, and I'd never store it at higher temperatures or in a fridge, because the times are then days, weeks or months. Bye -20 degrees you avoid a slow, gradually degradation, but keep a more or less constant concentration of active enzyme.

Edited by hobglobin, 22 November 2011 - 09:27 AM.

[tfitzwater]

Taq polymerase is quite stable at room temperature for six months providing it isn't opened!  If it is repeatedly opened you stand a very good chance of culturing some nice airborne bacterial or fungal contamination in the 50% glycerol storage buffer.
(Duncan Clark, DNAmp Ltd. (Licensed PCR enzyme manufacturer) on 03.08.96 at http://www.bio.net/bionet/mm/methods/1994-...ber/019005.html)

[little mouse]

I forgot Taq on the bench overnight.
It still worked.

It is better to keep it at -20°C, but if once you forgot it, you don't have to trash it. try with one or two samples to see if it works.
If I were your boss, I would not panic, but still I would shout on you not to forgot the enzyme on the bench.