Saturday, 6 October 2012

Restriction digest with OLD enzyme

I suspect most labs are like mine, with freezers overflowing with old, unused restriction enzmyes, bought for some odd bit of cloning, used once, then kept in the freezer forevermore.

In most labs I've worked in, the general rule of thumb for out of date enzymes was just add a few more units (up to the 10% of total volume permissable to prevent glycerol inhibiting the reaction), and maybe run a bit longer.

However, it's not until my current lab that I've ever found REALLY old enzymes. As in, enzymes older than some of the younger PhDs in the lab.

I've always suspected that the expiration dates on these are largely guesswork, so I thought I'd try out the oldest enzymes I could find.

I wanted to go with an aliquot of AluI I found, dated for September 1989 (although it doesn't say whether this is an expiry date or when it was made). Unfortunately, the lid for AluI is lost to the mists of time; the tube is just full of (likely 20-year old) ice.

However, I did find a tube of BstYI that appears to be of a similar age, perhaps even older; the labelling is fairly similar, but the lack of a date makes me question whether it predates the AluI (and inclusion of estimated expiration dates). Maybe there's someone at NEB who can sort this out - I'll investigate that. At the very earliest, it seems BstYI would have appeared around 1988.

I mixed up a quick 20μl digestion mix, using 2μl enzyme in NEBuffer 4 to digest around a microgram of pUC19 plasmid DNA, incubated at 65°C for two hours.

Lo and behold, it works just fine (presumably - I don't have any in-date BstYI to compare this against). This is an enzyme that is likely somewhere in the region of 25 to 28 years old. I'm not sure if I'll ever pay attention to expiration dates again.

NB: doing a quick search around for this write up showed me this article, which makes me wonder if I shouldn't be surprised; it implies that the companies don't tend to make up many batches of these enzymes, just thaw old ones and assay them for activity!

It's also probably worth noting that this seems to be a fairly robust enzyme, which has been kept at minus twenty for the vast majority of its life (it looks barely used), thus these findings might not apply to other enzymes.

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