In the event of having too much background in the transformation step, it is important to determine whether this is due to inefficient dephosphorylation or if it is due to undigested plasmid that has been carried over from the restriction digest. To establish the source of the background, two control samples need to be added in the transformation step:
- vector without ligase, and
- dephosphorylated vector with ligase.
If the number of transformants present in sample 1 is large, this is indicative of the presence of uncut plasmid in the vector DNA.
If the number of transformants in sample 1 is very low, but the number of transformants in sample 2 are high, this is indicative of inefficient dephosphorylation.
If the dephosphorylation step is determined to be the problem, make sure that you add the recommended amount of phosphatase per amount of substrate recommended. Also, make sure that you incubate for the recommended time and temperature.
We recommended heat-inactivating the restriction enzymes prior to the dephosphorylation step, if the restriction enzymes used can be heat-inactivated.We also recommend heat-inactivating the phosphatase prior to the ligation step. If the phosphatase used cannot be heat-inactivated, then we recommend a column cleanup of the DNA prior to the ligation step. This can be achieved by using our Monarch PCR & DNA Cleanup kit (5 ug) (NEB #T1030).