Help! Direction of ligation

[Keith Leppard]

ialibova at ARTSCI.WUSTL.EDU ("Irina A. Libova") wrote:
>
> Hi!
> I am ligating an insert into a vector, using HindiIII site on both 
> ends.  And I need a specific orientation.  But the problem is that on 
> ALL of my clones I get the reverse orientation compared to the one I 
> need, instead of getting a random 50:50 distribution of orientations.  
> Has anybode had the same problem, and what can I do to change the 
> orientation?

The orientation you want may be causing slow growth without being 
completely lethal. Have you tried leaving the plates longer and 
examining any small colonies that come up late? And/or culturing plates
at room temperature?

Keith Leppard