(1) It is possible that salt residues in buffers or ethanol residues in Wash Buffer are not removed completely and thus affect the downstream reaction. In case of salt residues, wash the column twice with Wash Buffer. In case of ethanol residue, after washing with Wash Buffer, make sure that the flow-through is discarded and centrifuge the column at full speed for 3 minutes. If necessary, to centrifuge for a few minutes more to ensure complete removal of ethanol.
(2) Another possibility is that plasmid is denatured. Denaturation happens if incubation in PD2 Buffer has been for too long time. This can be visualized during electrophoresis that a band migrates faster than the supercoiled form. After PD2 Buffer is added, do NOT incubate for more than 5 minutes.
(3) There are still other possibilities that could lead to fail in restriction enzyme performance of extracted plasmid:
Enzyme Activity: most restriction enzymes are temperature sensitive, prolonged storage or usage past expiry date will decrease enzyme activity dramatically, hence causing partial or total failure of plasmid cutting.
Sensitivity: different strains of E. Coli will have varying sensitivity to dam or dcm methylation, if digestion sites were blocked by overlapping dam or dcm methylation during transformation, it will cause difficulty at the restriction enzyme digestion step.
Plasmid Concentration: high concentrations of final extracted plasmid will cause similar problems.
Supercoiled form: high quality plasmid extraction kits always get high yields of supercoiled form of plasmids, please refer to the instruction of restriction endonucleases that customer use.