self ligating vector woes

[Peter Ianakiev]

Hi Paul
There always will be background. Unfortunately you can't avoid that.  In my
work it is less than 1% of the total, and it is due to nucleases either in
the Ligase itself or from some other source.
You probably can inquire about some special grade "nuclease free"  ligase if
1% background is too much for you. Shorter ligation reaction is also an
option.
Regards:
Peter

Paul Shinn wrote:

>    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