Restriction Endonuclease Digestion: Optimal Conditions

Having established the importance of choosing the right restriction enzyme, you and Eric are ready to move on with the practical aspects of the digestion. Before starting the procedure, Dr. Greene gathers you both for a discussion on creating the reagents and optimal conditions for the experiment. 

Dr. Greene: Before we actually set up the experiment we need to discuss the reagents that we will be using and the optimal conditions for running the experiment. This includes considerations like pH and temperature. Do you remember the reagents and the optimal conditions for restriction endonuclease digestion of DNA with EcoRI? 

You: It is a rather simple experimental set-up, as far as I remember. We only need the DNA from the suspect, the restriction enzyme, a buffer solution and water. 

Dr. Greene: That’s correct! What about the conditions of the experiment?

Eric: Don’t we need to carry out the experiment at a pH of 7.5? This is what the buffer is for; and we need to incubate the mixture at 37oC. 

You: That seems right to me, Dr. Greene. Do you agree?

Dr. Greene: Great job, that is correct! Do you remember why it is important to run the experiment at this temperature and pH?