The following controls can be used to diagnose this problem:
1. Uncut vector to check cell viability and test the antibiotic
resistance of the plasmid. Plate on media and media plus antibiotic.
2. Vector cut and not ligated to check for uncut vector. Gel
purification of the cut vector (we recommend the Monarch DNA Gel Extraction Kit, NEB# T1020) may be required to remove the last 0.1% of
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 (FAQs available for T4 DNA ligase, NEB#M0202).