Curious How they do the vector - regarding pTZ57R cloning vector (Mar/30/2010 )

Dear All,

I am thinking if I was given a vector pTZ57R, and have to turn it into TA cloning pTZ57R/T (Fermentas vector) vector,
I have to use EcoRV (Eco32I) to cut it. However, EcoRV is a blunt end cutter:
5'...GAT^TAG...3'
3'...CTA^ATC...5'

My question is, how can we make a "ddT" after "GAT" and another "ddT" before "ATC" ( refer to the Fermentas InsTAclone manual, between 650 and 651 of MCS region)
I know we can make "a-overhang" in PCR fragment by incubate with Taq polymerase but i didn't know how to add "T-overhang"

Any idea?


InsTAclone PCR Cloning Kit #K1213, #K1214

There are protocols on this website for T-vector construction:

http://www.protocol-online.org/prot/Molecu...ctor/index.html

Oh thanks for the reminder....I didn't notice that...
Thanks again.