self ligating vector woes

Paul Shinn pshinn at mail1.sas.upenn.edu
Thu Apr 18 17:11:32 EST 2002


   Hello,

   We are directionally ligating inserts into a vector using Sfi to make
the cut.  We are using SAP to prevent any self ligation even though by
nature the 2 Sfi ends of the vector arms are not compatible by 3 bases. 
We do this because we have seen self ligation in the past.  Most of the
time our ligations are fine but we have run into a problem where the
vector is self ligating but not because of the 3 base overhang.  It seems
the overhang has been chewed back and then the vector has blunt end
ligated.  This is the only theory we have based on what we see from
sequencing the clones we thought had inserts.
   Our vector DNA is gel purified, resuspended in Tris and then 
frozen at -20 until it needs to be used.  There may be one freeze thaw 
after it is initially stored away and that may be where the degradation 
is coming from.  We can't add EDTA because it might kill the ligation.  
Any suggestions?

						Thank you, Paul




More information about the Methods mailing list