I have cloned one of my mutant gene constructs into pet28a and we confirmed the presence of insert on the vector backbone with colony pcr and plasmid pcr multiple times even expressed the protein but unable to get the insert released on plasmid digestion of the same. Can anyone suggest something for this?
Try each enzyme individually (along with uncut control) to see if maybe one of them is not actually cutting.
You could also try cutting with REs who's sites are further outside the cloning site.
It might also be that the plasmid population is mixed, even if only a few percent of the plasmids are correct you would get a positive signal in PCR but when looking at a digest the insert band might be invisible.
Thank you Dr. Michael for you suggestions
I would also like to mention that we have cloned that insert by cutting with those same enzymes before so I am doubtful regarding the thought of whether they are able to cut or not.
And I would definitely try single digestion at a time and check l.
It may be that one site was destroyed in the clone you picked. Every once in a while you just get a bad clone.
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