It is possible that salt residue in buffers or ethanol residue in the Wash Buffer is not removed completely, thus affecting the downstream reaction. In case of salt residue, 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, centrifuge for a few minutes more to ensure complete removal of ethanol. Another reason is that the plasmid is denatured. Denaturation occurs if incubation in PD2 Buffer is too long. This can be seen during electrophoresis. After PD2 Buffer is added, DO NOT incubate for more than 5 minutes.