Problem:

Question 1: Why is it necessary to heat-inactivate exonuclease I?

Question 2: Why is it necessary to conduct two rounds of PCR in our cloning experiment?

Question 3: Some of your classmates just realized that when setting up the initial PCR reaction, the master mix MMNP (containing the nested set of primers was used). Then, for the second "nested" PCR, the group used the master mix MMIP (containing the initial set of primers). Do you expect this series of reactions to have yielded the same PCR product(s) as if they had performed the reaction correctly?

Please read all the questions carefully and answer them.