Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Is re-sequencing needed for insert ligated into a second vector? - (Mar/05/2006 )

Dear all,

I cloned two genes in pCRII-Blunt-TOPO vector with Pfx enzyme in PCR amplification, and positive colonies are confirmed by sequencing. Subsequently, I ligated these two genes to pcDNA3.1 vector in order to generate a fusion construct, hoping to express a fusion protein. My question is that should I sequence the whole fusion construct in pcDNA3.1 vector? even though there is no PCR amplification involved?

Thank you in advance.


Well, that depends on your goals. If this were an important construct for a research project I cared about, then I certainly would. If this is a throw-away test, then it probably is correct.