vector ligation: exonuclease activity

Z.Huang huang.zhong at med.kuleuven.ac.be
Thu Dec 11 14:44:52 EST 1997


Dear bionetters,

One guy in our lab faced such situation. He ligate his PCR product into 
T-vector and screened out positive clone and then purified his plasmid. 
Then he perform digestion analysis of the plasmid with EcoRI which 
flanked around the insert. He should get digestion band with correct 
size. But this time EcoR I can't cut the site. I am wondering what's 
happed? We confirmed the plasmid indeed has correct insert by PCR so it's 
apparently something wrong happed on plamid EcoR I site. I heared from 
forum that it's possible that ligation reaction can result in exonuclease 
activity that can occasionally delete the EcoR I site. Is it true in our 
case? Any suggestions are very appreciated.

Huang zhong
huang.zhong at med.kuleuven.ac.be



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