FAQ: The number of colonies that do not contain an insert seems high, how can I tell if the CIP worked?

CIP is a very robust enzyme and it is rare for dephosphorylation not to go essentially to completion. Low numbers of colonies containing vector with insert may be the result of incomplete inactivation or removal of CIP, causing dephosphorylation of the insert during ligation. Background may also be the result of uncut vector.

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 uncut vector.
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.