four-way ligation help !!!
damartin at bioslave.uio.no
Fri Jul 5 04:36:22 EST 1996
In article <Pine.SUN.3.91.960705001021.10662A-100000 at bio02>,
Robot Boy <ed at bio02.bio.uottawa.ca> wrote:
-->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
-->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...
You could try this though if having the HindIII site in the primer is not
PCR as per normal. (I presume using Taq - if using vent then don't t-tail
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.
* David Martin, PhD - Post-Doctoral Research Fellow *
* Atherosclerosis and Thrombosis research group *
* Biotechnologisenteret i Oslo *
* Postboks 1125 Blindern, N-0316 Oslo, Norway *
* Tel: +47 22 95 84 54 Fax: +47 22 69 41 30 *
* http://www.uio.no/~damartin/ david.martin at biotek.uio.no *
More information about the Methods