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plasmid digest - restriction enzyme won´t cut (Oct/18/2004 )


I tried to digest my pBSK(-) vector with EcoRV but failed. No linearized vector band on my agarose gel, just supercoiled + circular (nicked) DNA. I used 20 units of newly bought enzyme in the appropriate buffer + BSA, digested for 3 h at 37 degrees - should be more than sufficient. The vector is easily and completely digested by HaeIII in 1h. If the vector DNA from a Maxi-Prep is poor quality, it shouldn´t be digested by HaeIII either, right? Methylation shouldn´t be a problem for EcoRV.
Anybody having an idea?



Are you sure that your plasmid is what yopu think it is?
How many sites for EcoRV is in it?