four-way ligation help !!!

Robot Boy ed at bio02.bio.uottawa.ca
Thu Jul 4 23:25:13 EST 1996


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...

thanks for your thoughts,

Ed Taboada
Dept. of Biology
University of Ottawa
Ottawa, Canada



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