Hi Mahbobeh. Can you clarify - what do you mean by 'insert'?

Are you referring to losing the plasmid or to a deletion of the insert in the plasmid?

Can lose the plasmid if you don´t add the antibiotic to the media (if for example your plasmid include an antibiotic resistance marker).

Thank you all for your replies.

I checked the insert using clony pcr and confirmed it. after 3 weeks when I recultured the cells and did miniprep, I have not seen the insert at all. it seems that either the plasmid or the insert are gone. the interesting thing is that the bacteri grow on ampicillin media. that is why I guess that probably my insert is gone.

I would just retransform your original plasmid prep and pick new colonies. However to answer your question, it is unlikely that the insert was selectively deleted unless there was a strong selective pressure against it (such as it was toxic) but had that been the case you would have had trouble with the original cloning.

thanks Michael,

fair enough. Yes i am doing transformation and hope it works this time.