PacI weirdness

brett at BORCIM.WUSTL.EDU brett at BORCIM.WUSTL.EDU
Wed Oct 5 20:27:17 EST 1994


Hello. Our lab has noticed a weird behaviour of NEB's PacI. When we first get
a tube of the enzyme, it works great. We store all our enzymes in a large
constant temp freezer @ -20C, in Stratacoolers. Over time, rather rapidly,
ie within a month, we see other nuclease activities  in the PacI. In a
controlled digestion, this usually means several extra bands, but in an over-
digestion, we're talking smear. The extra bands do not follow the pattern of
a partial digestion. Importantly, this even happens in UNOPENED, stored tubes
of PacI. I have called NEB, and they either think I'm sloppy or crazy. They
have even replaced a tube on request. But, no matter, this is a phenomenon
we have seen several times with different batches. I also hasten to add that
we do not see this with any other enzyme, and we are VERY paranoid about
maintaining our enzymes. We even have tubes of things from the mid-'80s that
still cut well and do not show extraneous nucleases. So, what gives with PacI?
Has anybody else seen this? NEB says no, but I thought I'd check with common 
knowledge around here. My hypothesis is that PacI is not real stable, and
maybe it breaks down upon storage and/or transport, changing the relationship
between recognition activity and scission activity (ie the PacI has different
specificity). Any suggestions? Please help, as we could go broke buying new
tubes of this stuff constantly.

brett at borcim.wustl.edu

p.s. To test my hypothesis, I thought of looking at the PacI on a protein gel,
but haven't actually gotten around to doing so. Other suggestions?



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