self ligating vector woes
ianakiev at genome.wi.mit.edu
Mon Apr 22 12:37:26 EST 2002
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
Paul Shinn wrote:
> 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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