Restriction Digestion - problems (May/07/2004 )
I am currently trying to carry outa fairly simple double digestion of a standard yeast plasmid however, the vector does not seem to cut.
apart from the obvious probelms of enzymes and buffers can anyone suggest anything else to improve efficiency,
Pay attention in temperature and time of incubation, SDS in reaction or any other like this. Try with the same enzyme, but with DNA, no with the plasmid.
By, and good luck
It would be easier to answer your question if we knew which enzymes and which restriction buffer(s) you are using. How many units, how much plasmid, plasmid prep method, time and temperature of incubation? Do the enzymes cut a plasmid prepared elsewhere? Does that plasmid cut if you mix it with your plasmid to check for the presence of inhibitors in your plasmid prep?
Assuming the right buffers are being used in the right sequence for the double digestion, it is possible that certain non-specific DNA binding proteins might be locked on to your plasmid. Try a phenol extraction on the sample before repeating the digestion
If you use only one restrict enzyme, can the vector be cut?