Hi,

I want to insert six piece of gene in the pBAD30 vector. Those genes were previously cloned into pHRED30T vector ( between NcoI and HindIII restriction sites ). For this work, I chose primers from the flanking region , amplify the region, purify the PCR products and then cut with BamHI and HindIII. Noted, the flanking region contains BamHI and HindIII sites within it. I also cut the vector pBAD30 with same enzymes. In pBAD30, BamHI and HindIII is 80 bp apart. So, when I run in the gel, I saw double digested product and single digested ( either cut with BamHI/HindIII only) product gave similar sized band. However, after overnight ligation by using T4 ligase ( I didn't use alkaline phosphatase) I did the transformation. I got ~10 colonies for the ligated product of vector and insert, no colonies for double digested vector with no insert . And, ~200 colonies for pBAD30 plasmid. Seems transformation worked. To screen, I miniprep some of the positive plasmids and digested with BamHI and HindIII. I also digested the pBAD30 plasmid. In the gel, I saw only band similar to double digested vector. No band for the insert. My inserts are 180 -500 bp. Where did I make a mistake? How I can troubleshoot this cloning?

Thanks