Lack of colonies after transformation is often not indicative of a failed ligation. Competent cells vary greatly in their efficiency. The use of high efficiency competent cells can make a dramatic difference in the perceived success of a ligation. Also, the integrity of the DNA used can strongly influence the result. For example, a minute amount of exonuclease present in a DNA sample prior to ligation can chew back an overhang and prevent compatible end ligation. Additionally, salts left over from purification steps can inhibit or decrease ligation efficiency.

Other factors affecting transformation include the requirement that DNA ends to be joined must be compatible with at least one partner containing a 5’ phosphate. DNA generated from PCR needs to employ phosphorylated primers during synthesis or be treated with a kinase after synthesis. DNA prepared by restriction enzyme digestion maintains 5’ phosphorylation. Blunt ends can be joined to any other blunt ends, while fragments with overhangs need to base pair correctly to be ligated.