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.