I digest my plasmid with KpnI-NotI. I cut the expected 4kb band from the gel, purified it and then I did ligation [1:3 and 1:5 vector:insert ratio] and transformation. I found 18 transformed colonies in total. I grew the transformed colonies in LB broth, isolated the plasmid and performed KpnI-NotI digestion again to confirm the presence of my 4kb insert in the plasmids from transformed colonies. 4kb and 5kb were my expected bands on the gel. Unfortunately, none of my colonies carry expected 4kb band. Some carry empty vector, others carry band smaller than 4kb. I have clue, what is happening. Any suggestion?