I tried to clone a 750 bp insert in a 5 kb vector, I did it the old fashioned way as following:
Cut the vector and insert using ECON1 and Nru1. Dephosphorylation of the vector using the CIP, transformation, I got many cultures. after that I amplified the miniprep using insert specific primers and all looked good on the gel. after DNA sequencing I found that the vector has my insert but it was cloned inverted into the plasmid !!!!! Does anyone have an idea why did this happen???