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  • FAQ: The number of colonies that do not contain an insert seems high. How can I tell if rSAP worked?

    rSAP is a very robust enzyme and it is rare for dephosphorylation not to proceed essentially to completion. The following transformation 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 (we recommend 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, dephosphorylated and ligated: to check for dephosphorylation. Total colony number should be <5% of the vector cut and ligated.