I am pretty new at this, I am trying to order my gene of interest, full cDNA construct from Genscript, and trying to ligate that to pcDNA3.1 directional V5-His-Topo vector from invitrogen. This is to simplify matters and not having to do the PCR/cloning for the construct. Genscript is placing the insert in puc57 vector. But the pcDNA3.1 directional TOPO vector kit includes the PCR step for which to enable directional cloning, the forward PCR primer must contain the sequence, CACC, at the 5′ end of the primer. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in pcDNA™3.1D/V5-His-TOPO®.(see attachments)
So I am unsure on whether I need to add the CACC to my ordered gene construct?
If its placed in puc57 vector and I have to digest using restriction enzyme to isolate the insert for ligation, won't the ends be different from CACC required and cause the ligation to directional TOPO to not work?
Please help, will appreciate any advice. Thank you