Which step can reduce empty vector background?

Correct answer: Vector dephosphorylation

The governing concept is prevention of vector self-ligation during cloning. After a restriction enzyme cuts a vector, its compatible ends can sometimes join to each other without an insert, producing an empty vector that generates background colonies. Treatment of the cut vector with an alkaline phosphatase removes the 5-prime phosphate groups needed for efficient ligation of the vector ends. The insert can still be ligated when its phosphorylated ends provide the required phosphate. Thus option A is correct because vector dephosphorylation lowers self-ligation and consequently reduces empty-vector colonies. Water, routine host removal, or boiling the insert does not provide this specific control and may damage the cloning reaction.