Does CIAP cause ligation problems?

Steven Goldberg goldberg at bms.com
Fri Jan 10 08:04:37 EST 1997


In article <5b39st$6qv at info.service.rug.nl>, g.j.e.j.hooiveld at farm.rug.nl
(Guido Hooiveld) wrote:

> Hi everybody,
> 
> For the last two months I am trying to perform a non-directed ligation. 
> Therefore I tread my vector with CIAP (Promega). However, till now I wasn't 
> successful.
> 
I gave up on CIAP a long time ago because of the difficulties I had
getting fragments to ligate.  I think there is a lot more exonuclease
activity associated with this enzyme than manufacturers are willing to
admit.  Anyway, the best alternative for routine subcloning or plasmid
library construction has been pZeroI by Invitrogen, where I routinely get
90% recombinant plasmids.  If I have to go into some sort of specialized
vector, then I will flood the vector with insert (5:1 or 10:1 ratio)
and/or use directional cloning wherever possible.  In these cases I
usually end up with ca. 20% recombinants.

Steve



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