Ligation conditions should also be checked (see FAQs available for T4 DNA ligase, NEB #M0202 ).
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: 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, treated with CIP and ligated to check for dephosphorylation; should be 3-5% of the vector cut and ligated.