four-way ligation help !!!

[David Martin]

In article <Pine.SUN.3.91.960705001021.10662A-100000 at bio02>,
   Robot Boy <ed at bio02.bio.uottawa.ca> wrote:
-->Hi all,
-->
-->I need a little bit of help/advice. I need to clone the coding region of 
-->the gene I'm studying into an expression vector which can only accept 
-->inserts in an NcoI to HindIII orientation. I'll be introducing the 
-->restriction sites via primers which I'll use to amplify the coding region.
-->
-->My problem is that the coding region does contain internal HindIII sites.
-->I'm thinking that perhaps the way to go would be to just do the 
-->amplification, cut the PCR product (or the cloned PCR product) with 
-->HindIII and Nco I, and just do a 4-way ligation of the three resulting 
-->fragments along with the NcoI/HindIII cut vector, followed by PCR screening 
-->of my transformants using primers which could let me figure out the 
internal 
-->arrangement of the HindIII fragments, hoping that one of them will have 
-->the proper sequence of HindIII fragments (did this make sense ?)
-->
-->Now...as I scramble to ponder how to pull this off, this seems as good a 
-->strategy as I can come up with, given my present low-caffeine 
-->status...anyone with thoughts/suggestions to steer me in the right path ?
-->Should I be more methodical in doing this ?  I'm sure *someone* has had 
-->to deal with this in the past...please help me...and I'll buy you a nice 
-->slice of chocolate cake...
-->
Umm.. Tricky. 

You could try this though if having the HindIII site in the primer is not 
critical..
PCR as per normal. (I presume using Taq - if using vent then don't t-tail 
below).
Cut the fragment with just Nco 1

Cut the vector with HindIII and then blunt end it
 followed by T-tailing (Marchuk,D et al, NAR (1991)).

this gives you a T-vector.
Now cut the vector with Nco 1 to leave you a T-overhang on what was the 
HindIII site and an Nco 1 site the other end.

So you now have two cohesive ends and can screen for recombinants by PCR.

hope this helps.

.d

* David Martin, PhD -  Post-Doctoral Research Fellow        *
* Atherosclerosis and Thrombosis research group             *
* Biotechnologisenteret i Oslo                              *
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