four-way ligation help !!!
john.brennand at gbapr.zeneca.com
Fri Jul 5 07:37:57 EST 1996
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
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
Hope this helps
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