Is competent cell still good in -80oC for 16 months? - got a different plasmid after plasmid prep (May/23/2006 )

I transformed 1ug plasmid into competent E.coli DH5alpha cells. The transformed cells were seeded first into 2ml then 50ml LB+amp, cultured ovenight and the cells were lysed and plasmids were purified using Bio-rad kit.

The problem is: I sequenced both the original and the newly prepared plasmids, and they have 1bp difference. And unfortunately the different bp is exactly the phosphorylation site that I want it to be exactly right. I don't known what's wrong with such a simple plasmid prep. The only thing I can think of is my competent cells are 16 month old so that I'm not sure whether it's good or not. I'm stucked. Any help?

I havent had problem with such old electrocompetent cells. Try to retransform them in new cells if it helps.

Just curious , y did u use 1ug of DNA to transform?

Thanks for your advise. I'll try to make some fresh competent cells tommorrow and try again next week.