self ligating vector woes
wolfsc at ibms.sinica.edu.tw
Thu Apr 18 20:39:32 EST 2002
If you get blunt end ligation, there is some DNAse present probably in both:
vector and insert (maybe in the SfiI vial?)
Unless you generate a library: if the fragmet you cut out of your vector is
small, just do a column cleanup or EtOH precipitation. Sicne the small
fragment is dephosphorylated, I wouldn't expect too many re-assembled vectors
in your ligation.
> 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
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan
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