There are a few possible reasons that could lead to failure in restriction enzyme performance of extracted plasmid. (1) 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. (2) 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. (3) Plasmid Concentration: High concentrations of final extracted plasmid will cause similar problems.