The following controls can be used to diagnose this problem: 1. Uncut vector: to check cell competency and antibiotic resistance. 2. Vector cut and not ligated: to check for uncut vector, gel purification may be required to remove the last 0.1% of uncut vector. 3. Vector cut and ligated: to check for intact ends and ligation conditions. 4. Vector cut, phosphatased and ligated: to check for dephosphorylation, should be 3-5% of the vector cut and ligated. Ligation conditions should also be checked.