DNA Digestion

• 1). Add a restriction enzyme to your DNA sample. Use EcoR1, a commonly used enzyme, for example.
  
  
• 2). Add this mixture to a buffer solution, which is essentially a place for the reaction to occur.
  
  
• 3). Raise the temperature of the reaction, as indicated in the New England BioLabs manual. Each restriction enzyme has a specific reaction temperature at which it functions best. Additionally, each enzyme has a specific nucleotide sequence it is designed to target. EcoRI will scan and cut the DNA at the nucleotide sequence CAATTC. This exact order of nucleotides must exist for the enzyme to bind and cut the DNA. Until this point, all materials should be kept on ice to prevent unwanted activity.
  
  
• 4). After allowing the reaction to occur as indicated in the manual, run the mixture in a gel electrophoresis machine to separate the DNA fragments based on size. The smallest fragments will move the farthest across the gel.
  
  
• 5). Measure the distance traveled by each DNA fragment. These five steps should be repeated with multiple different enzymes separately.
  
  

Constructing the Restriction Map

• 1). Using the data obtained from the gel, collect all the information you have found using the different restriction enzymes.
  
  
• 2). Deduce the order of the restriction sites by comparing the different fragment lengths produced by each enzyme. For example, if enzyme #1 produced two fragments of equal length and enzyme #2 produced three fragments of equal length, you can conclude that enzyme #1 cuts halfway through the DNA and enzyme #2 cuts the DNA into thirds.
  
  
• 3). Using the conclusions drawn in step 2, assemble the order of restriction sites for the DNA.