I am using Exonuclease I for its ss 3' - 5' exonuclease activity to clean up after second strand synthesis. I immediately follow up with T7 exonuclease digestion (ds 5'-3' exonuclease activity) to remove the first strand and leave behind the second strand product for downstream work; the second strand is unaffected during T7 digestion due to the addition of a 5' adapter sequence, effectively leaving the 5' region hang as a ss 5' end.

In trying to reduce complexity in my workflow, I am asking if anyone has tried, has knowledge of, or can give insight into whether it is possible to add both enzymes in a single reaction pot (they both work in NEBuffer4) but have them work independently by switching the reaction temperature. I am looking to preserve my second strand product without having an a somewhat active Exo I partially digest the second strand product.