4 way ligation avoided !!!!!!

Robot Boy ed at bio02.bio.uottawa.ca
Fri Jul 5 14:45:48 EST 1996


Once again, you've all proven to me that the internet is not just a tool 
for obtaining cyber-smut...

Thanks to all who answered regarding my little cloning dilemma.
I think it has now been resolved (thanks David !)

I'm forced to clone an NcoI/HindIII fragment [amino to carboxy] because 
of the vector I have to clone into, and there are two internal HindIII 
sites. David suggested perhaps exploiting unique sites on the HindIII 
fragment that is at the carboxy end. I will most likely use a KpnI site.
 
if my PCR product is :

N-term 						      C-term
NcoI---------HindIII--HindIII---------KpnI-----------HindIII
 

I will then do two digests of the PCR product (which has been amplified 
with NcoI and HindIII bearing primers). A KpnI and HindIII digest should 
yield the KpnI/HindIII fragment and an NcoI and KpnI digest should yield 
the NcoI/KpnI fragment. 
A three way directional ligation of NcoI/HindIII cut vector, 
NcoI/KpnI fragment, and KpnI/HindIII fragment should then yield the 
appropriate construct if all goes according to plan.
 
Thanks to all. You've been of great help :)

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



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