four-way ligation help !!!

[John Brennand]

I think the blunt ending option is best - 3 & 4 way ligations are 
pretty inefficient and Sod's law says that you will get every 
combination of fragments bar the one you want.

Direct clone the pcr product into a T vector (or a blunt cut vector 
if not using Taq) that has a blunt site, that doesnt cut your 
insert.

Identify a clone in the correct orientation and release the insert 
with Nco & the vector blunt end site (which will be downstream of 
your stop codon).  Clone it into Nco - blunt expression vector.

Alternatively, cut the pcr clone to completion with Nco to linearise 
it and then do a partial HIII digest on ~ 10ug of this DNA. This 
will give you a spectrum of bands but you know the exact size of the 
one you want so you should have enough of it to then isolate off a 
gel.

Hope this helps

john