This page was created as an assignment for an undergraduate class at Davidson College.
How to run restriction digests
(Protocol taken from Campbell, 2002)
First you need to decide which restriction enzymes you are going to use. Pick enzymes that are at your disposal and that cut both the plasmid and insert once. If possible try to use only one restriction enzyme. Each restriction enzyme works best at an optimal pH and salt concentration. When the enzyme is not run under optimal conditions star activity can result. Star activity is when the enzyme loses its specificity. The optimal conditions are controlled by the buffers used. If using two enzymes, try to use enzymes that use the same buffer or have 100% or near 100% activity for the multi core buffer. You will have to use a table to find out what buffers and conditions are needed for each enzyme. After you decide which enzymes and buffers you are going to use you can design the digest.
Things to consider when running restriction digests:
-Restriction enzymes come in a 50% glycerol solution. You never want more than 5% (v/v) glycerol in a restriction digest.-Buffers usually come as a 10 x stock-You never want the restriction enzyme to be more than 10% final volume
Example of a digest that will have a final volume of 20 ul:
5 ulDNA2 ulbuffer (10x)0.2 ulBSA1.0 restriction enzyme (<10%)11.8 ulwater__________________20 ul total volume
Make a mix of everything but DNA. Remember to add the restriction enzyme last. Then add the DNA. Run reaction for at least one hour at optimal temperature (usually 37 degrees Celsius).
You have just digested your plasmid!