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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:

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

-adrian kohsf-

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


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

-adrian kohsf-