Hello seniors,

The suicide plasmid pRE112 was subjected for gene knockout construction. However, after several attempts the negative control (linear vector only) plus tests groups (linear vector + insert) plasmid transformation have abundance growth on media plates indicating failure of the construction. According to the kit instruction, the negative control growth indicates false positive colonies due to failure of vector linearization. Furthermore, the gel results confirming its linearization and previously several plasmids were successfully constructed by the same way. Where I am making the mistake?

Looking Forward,