Restriction Digest of pGLO plasmid

Aaron BautistaHongngan Van, Jesse MartirBiology 210ASan Diego Mesa CollegeJuly 13th, 2015

Methods Microcentrifuge tubes, a marker, a P-10 and P-20 micropipette with disposable tips, a sample of pGLO plasmid DNA, the particular restriction enzyme digests for this lab and their respective buffers, a minicentrifuge, and a water bath set at 37 degrees celsius were gathered in preparation for the experiment.Restriction enzymes Eco RV, Pst 1, and Eco RV + Pst 1 were placed in three separate tubes along with its respective buffer, and were then pipetted with plasmid DNA. After the tubes were centrifuged, each tube was set in a 37 degree water bath for 45 minutes.Place the DNA fragments that resulted from each digest onto a 1.0% agarose gel, and observe each DNA fragment as they travel towards the positive electrode. Before the DNA samples are loaded from each digest into separate wells on the gel, make sure the DNA samples are mixed with a loading dye. The agarose gel was mixed with ethidium bromide (see details in the lab manual regarding how to properly handle ethidium bromide). After electrophoresis was finished, the ethidium bromide stained DNA bands were observed and the gel was photographed as a permanent record of the DNA bands.A gel-casting tray was prepared while a well-forming comb was inserted. The agorose gel was casted by the use of a bottle of molten 1.0% agorose gel and 5 l of ethidium bromide then placed in the gel casting tray to solidify for approximately 10-15 minutes. The resulting gel was then prepared to be loaded in the electrophoresis chamber, which was filled with electrophoresis buffer.6X loading dye was added to the three digestion tubes, along with the DNA ladder. After loading dye was added to each tube, a total of four tubes were spun in the centrifuge and then each sample was pipetted into its own well.The samples were prepared to be run. Orient the electrophoresis chamber as shown in Figure 8c in the lab manual, and run gel at 100v for 30 minutes to an hour.