Directional cloning 2bps overhang - (Jun/04/2014 )

Dear all,

would a 2 basepair overhang be enough for directional cloning? I would like to use BstBI , giving a GC overhang and a CG overhang.

(TT'CGAA)

(AAGC'TT)

Is this enough or should I try to go for a bigger overhang?

It should be fine - how big is the other end?  If the ends don't match then you should be fine.

I am not sure what you mean? I cut with 1 restriction enzyme, to open my vector, and I will have a CG overhang and a CG overhang.

its like this:

TT             CGAA

AAGC            TT

So I have the GC and CG overhang and in those I want to put a fragment.

Ok- that's not directional cloning as your insert can go be inserted in either direction.  For directional cloning you need to have two different restriction ends so that the insert should only insert in one orientation.  Yours will still work, but you need to screen for direction.