DNA Separation By Electrophorosis

DNA, Deoxyribonucleic acid, is a double stranded, helical nucleic acid molecule
which determines inherited structure of a protein. The "steps" are made of
bases: adenine, guanine, cytosine, and thymine. The sides are sugar and
phosphate molecules. Restriction enzymes are enzymes that cut DNA at restriction
sites, leaving fragments blunt or sticky. The restriction fragments are
separated using a technique called gel electrophoresis. DNA has a negative
charge so when an electrical charge is applied it makes DNA move to the positive
side. DNA is placed in agarose gel. Smaller fragments move faster. The purpose
of this lab is to separate DNA fragments using gel electrophoresis. Hind III
cuts AAGCTT between the two irst A’s. EcoRI cuts at GAATTC between the G and
the A. Hind III and EcoRI both make sticky ends. Results: Our results for this
lab were EcoRI separated into five fragments. Hind III separated into four
fragments. The control only had one fragment. (See chart A and figure 1-1 for
distances) Discussion: The purpose of this lab was to see how gel
electrophoresis separates DNA fragments. We used Hind III, EcoRI, and a
controlled enzyme. Some fragments were hard to see because of smearing. These
were the bigger fragments. Loading the DNA was difficult and if you weren’t
careful you could rupture the wells which ruined the lab. We, fortunately, did
not run into this problem. Abstract: The purpose of this lab is to separate DNA
fragments with gel electrophoresis using EcoRI and Hind III. Restriction enzymes
are used to break up the DNA, then negatively charged DNA is placed in a gel
casting tray. Then it is placed into an electrophoresis chamber. An electrical
field is placed across the agarose gel which forces the fragments to move down
the gel. The amount of lines show how many fragments there is in the DNA. We had
five fragments for EcoRI and six for Hind III. The no enzyme had only one
fragment. Procedures: We sealed the ends of a gel casting tray with masking tape
and inserted a comb into the slots. The tray was filled about 6mm high with
agarose gel. It covered half the height of the comb. We waited ten minutes for
the gel to solidify. Then we placed the tray in a gel box and made sure that the
comb was at a negative (black) end. The box was filled with tris-borate-EDTA
buffer so it covered the entire surface of the gel. The combs were removed
without ripping the wells. The micro pipet was used to load the lambda EcoRI,
lambda Hind III, and lambda only into the wells. We dipped the pipet trough the
surface of the buffer over the wells and expelled the contents. The top of the
electrophoresis chamber was closed and electrical leads were connected. The dye
was observed as it moved shortly after the power supply was turned on. The power
supply was turned off after the bands migrated near the end of the gel and the
top of the electrophoresis chamber was removed. We removed the gel from the gel
casting tray and examined it under a light box and compared it to the ideal gel


References: Restriction Enzymes: Cleavage of DNA lab University of Illinois.
(1999). Experiment 2 Gel Electrophoresis of DNA. In Molecular Biology Cyberlab,
online: Http://www.life.uluc.edu/molbio/geldigest/electro.html