digestion with pvu1 enzyme - (Sep/16/2005 )
can i know whether enzyme which is 4 years old can still be functioning or not?im using Pvu I from amersham and it supposedly yield only one band,but unfortunately i got 3 bands instead of one...
If your DNA is all right, I guess it's star activity. Check it first.
Or the enzyme is contaminated with nuclease if the bands contain smear too.
Well, too old (=inactivated) enzymes hardly cut DNA.
I have used 11 year old restriction enzymes without any problem. Enzymes tend to lose activity rather than act indiscriminately. BamH I is prone to aggregation and may not last as long as other restriction enzymes.
Pvu I is not listed by New England Biolabs as having star activity.
Try the Pvu I on another similar plasmid to see if it is your DNA prep.
There is the risk that someone else has contaminated the tube of Pvu I with another restriction enzyme by using the same tip twice.
Your plasmid map may be in error if this is a construct rather than a commercial vector.
An alternate explanation of what you're seeing may be that the enzyme has no activity -- the three bands are the three different forms of plasmid DNA -- supercoiled, nicked circular, and linear...
It's tough to say if this is what you're seeing without seeing the gel. You could run an undigested sample of the DNA in an adjacent lane and compare.