A couple of questions about controls you are running during your procedure:
(1) Have you checked to see that your enzyme is indeed cutting the plasmid?
(2) Are you dephosphorylating the plasmid to help prevent self ligation?
(3) What is the molar ratio of insert to plasmid? It should be about 4:1
(4) How do you purify your insert? Gel purification or something else?
These are simple things, but from the info you gave, I'm starting from
scratch.
Jay Mone'
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