How can I be sure that the restriction enzyme digestion is complete?
The protocol uses an excess of each restriction enzyme and a sufficient incubation time relative to the recommended DNA amounts. This combination is more than sufficient to completely digest the DNA sample. The larger the difference between the Ct values of the mock and double digests [W = Ct (Mo) Ct (Msd)], the more complete the restriction enzyme digestion. Because Ct values are inversely and exponentially related to the initial amount of DNA material, each unit of cycle difference between these digests represents an additional two-fold difference in the amount of DNA. For example, a five-cycle difference means that the double digest contains only (100 x 2 ^ -Ct = 100 x 2 ^ 5 = ) 3.125 percent of the DNA of the mock digest, meaning that the reaction is therefore (100 – 3.125 = ) 96.875 percent complete.
