RE Digest

     A Restriction Enzyme digest involves cutting up a piece of circular DNA. Seems simple, right? The problem is, contamination is very common in RE digests, so it is important to know exactly what you are doing, and how everything works.

Background

     Restriction enzymes are isolated from bacteria. They recognize specific sequences of DNA and cut it to produce fragments. Usually, scientists use restriction enzymes to track where the restriction sites are on a certain plasmid or DNA strand. Scientists have isolated enzymes from their bacteria so that they can be used in the lab. In this specific RE digest, we will be using EcoRI and PvuII to cut PGBR22, a plasmid from Montipera efflorescens that codes from a purple fluorescent protein.
     To perform the digestion, obtain a tube each of EcoRI and PvuII, the plasmid you wish to cut (in this case PGBR22), 10X Enzyme buffer (make sure this is a suitable buffer for both enzymes so that they can function at their maximum efficiency), water, and three empty, sterile tubes. In each tube, place 1.5ug of the plasmid (figure out how many uL of the plasmid you need using the concentration) and 2.5uL buffer. In the first tube, put 1uL EcoRI; in the second, 1uL PvuII; and in the third, 0.5uL of each enzyme. Then, calculate how much water you would need to make a total volume of 25uL and add it to each tube. Pipette the sample up and down to mix and spin the sample down in a centrifuge.
     Next, incubate the samples in a water bath at 37 degrees for about 1.5 hours. This will allow the enzymes to do their jobs: cutting the plasmid. After time is up, spin down the samples once more, and place them on a heat block at 80 degrees. This step will denature the enzyme so that it will not function anymore (you do not want the enzyme to keep cutting the already cut plasmid).
     That's all there is to the digestion part. Next, you will have to check if your digestion worked. See the next post on gel electrophoresis!