XhoI digestion - (Sep/18/2011 )
here is the composition i usually set up:
1- 0.5-1 ug of plasmid DNA 5ul
2- 10X restriction enzyme 2ul
3- XhoI 1ul
4- D.W 12ul
Incubation: 60-90 min at 37 centigrade degrees
I have tried other restriction enzymes and they work properly, so, i assume the DNA is okie, I have asked colleges of mine to use the same batch of XhoI I used, it works on their fragments, then the enzyme is okie, i suppose.
I would say that either the cut site you are trying to cut is not there, or that it is methylated, preventing it from being cut. But XhoI is not sensitive to the normal E. coli methylases. This leads me to believe your cut site is simply missing. Have you sequenced the region?
to my surprise XhoI recognition site was exactly there in sequencing results and even using JM110, Ecoli strain with no dam/dcm system, it was still impossible to cut the site. pretty strange!