self ligating vector woes

[Wolfgang Schechinger]

Dear Paul,
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.

Regards,
Wo

>    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
> 
> 


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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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