After 17 years of sub-cloning plasmids etc something strange happened last week:

My initial cloning plan involved cutting a plasmid with Cla I and another enzyme. The initial test digest showed that ClaI did not cut. Since the enzyme was 4 years past its use by date (!) I quickly ordered a new one - which didn't cut either. OK, so ClaI is probably methylated - ordered in some methylation negative bugs.

After doing the transformation and maxi plasmid prep, I repeated the digests - and ClaI cut. Later on in the cloning plan a digest gave an unusual result. This turned out to be because a single cutter that was supposed to be in the plasmid was in fact not present. OK new plan required.

The new plan was a rather unsatisfactory one which involved blunt cutters and blunting sticky cutters with a mixture of filling in and "chewing back".....but as I'd already lost enough time I just got on with the blunt-blunt plan.

I digested the plasmid (the one that went through the methylation negative bugs) with PvuI and SmaI. SmaI DID NOT CUT and for PvuI there was an EXTRA CLEAVAGE SITE!

After giving the subconscious the weekend to mull over the problem, I re-tested the plasmid with each enzyme invividually. I also tested the original plasmid (before it went through the methylation neg. bugs). This showed that the original plasmid cut exactly as expected whilst the CH3- plasmid again failed to digest with SmaI and produced an extra band with PvuI.

Pretty damned strange.