• My NEB
  • Print
  • PDF
  • 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. Ligation conditions should also be checked (see T4 DNA Ligase). Background may also be the result of uncut vector.

    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, CIPed and ligated: to check for dephosphorylation, should be 3-5% of the vector cut and ligated.