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
(figure1-2).
Bibliography
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