subcloning into bacterial expression vector (View forum version)



Janesh Gautam

Posted 22 January 2011 - 07:36 AM

hi freinds
i want to subclone my insert(1.5kb) from pcdna3 to pET series or any other bacterial expression vector with N or C terminal His tag ,for efficent expression & purification of my insert & then do its structural analysis by CD.my problem is that my gene was cloned in pcdna in hindIII & NotI but these site are collinear in pET vector series .can u suggest any alternative

bob1

Posted 22 January 2011 - 12:45 PM

You could cut out the insert using the RE's you mentioned, and then ligate on adapters with RE sites that will allow you to clone into the bacterial expression vector.

You could PCR the insert with restriction sites on the primers and then clone from that.