4 way ligation avoided !!!!!!
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 :
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 :)
Dept. of Biology
University of Ottawa
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