PmeI-a mind of it's own? - (Sep/17/2007 )

I'm having trouble getting PmeI(New England Biolabs) to cut certain plasmids. I've used it successfully in the past and others in the lab have as well, but when I try to remove an insert out of a particular construct, it seems virtually inactive. I've tested old as well as new stock. I see faint bands on a gel after a digestion, whether for two hours or overnight. Is there something I'm missing as far as methylation or something? I isolate my cDNA constructs from JM109. This sequence verified PmeI site is in the the context:

...GCTAGCGTTTAAACTGTACA...

I've emailed NEB as well, but haven't heard back. Is anyone else having trouble with this RE?

Hmm... just wondering... might you have sequence your cDNA? Could there be a mistake during the cDNA synthesis?

And just checking, the DNA is clean enough for digestion, right?

JM109 is EcoKI methylation +, and PmeI is sensitive to EcoKI methylation. I can't tell from your sequence, but if it is GTTTAAACNNNNNNGTGC or GCACNNNNNNGTTTAAAC, then methylation will occur and cutting will be impaired or inhibited. See rebase.neb.com and look up PmeI, then check for methylation sensitivity. You could clone into DH10B or Top10, which are EcoKI methylase -. See http://openwetware.org/wiki/E._coli_genotypes for genotypes and look for hsd(M-).

One in 256 for each case; one in 128 for either. This assumes you have 50% GC content in your genome DNA.