Restriction Digest of pGLO plasmid

Aaron Bautista
Hongngan Van, Jesse Martir
Biology 210A
San Diego Mesa College
July 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.

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