Dna Gel Electrophorosis Essay, Research Paper
Introduction:
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