Light blue colonies or really blue colonies obtained using the QIAGEN PCR Cloning Kit
can also be positive clones. Sometimes, the LacZ alpha fragment and the cloned insert are in frame, resulting in a protein with beta-galactosidase activity. As this protein might not be very active and the X-gal may not be digested as rapidly, colonies may appear only light blue. The likelihood for this to occur increases with larger inserts.