problem with restriction digestion - (Jun/02/2007 )

Hi
I am trying to clone a 1 kb pcr product within Xho 1 and Xba1 sites of pET 32 a vector. The vector was isolated from BL21DE3 strain. I found that Xba1 is not cutting the plasmid properly. Can it be due to dam sensitivity?.My vector sequence is tccccTCTAGAaat.( Xba1 recognition seq is in capitals and flanking seq in small case). Can my vector be resistant to Xba 1 digestion? Is it true that for Xba to be sensitive to dam methylation the recognition site tctaga should be followed by tc?. I am using NEB's enzyme which is stored at -20 C. The vial says assayed on 03-2005. How long do enzymes remain intact when stored at -20? Please help me out.

That XbaI site should cut. In general, working with DNA prepared from BL21 cells is problematic because of endonuclease contamination problems. I would recommend transforming a cloning strain (DH5a, Top10 etc.) with your plasmid and re-extracting from a miniprep done on one of those strains.

I have 2 vials of BssHII in the lab from 1998 and it still works fine. So if kept carefully, I guess the enzyme can last some time.

But perhaps you could do a trial and see if the XbaI can cut DNA derived from another source.