Distance

43 mm
48 mm
66 mm
75 mm
80 mm
94 mm

Eco RI
Cal. BP
20,347 bp
17,398 bp
9,901 bp
7,470 bp
6,387 bp
4,120 bp

Bam HI
Cal. BP
16,342 bp
13,973 bp
9,901 bp
8,736 bp
7,470 bp

Act. BP
Distance
24,756 bp 50 mm
21,226 bp 55 mm
7,421 bp
66 mm
5,643 bp
70 mm
4,878 bp
75 mm
3,530 bp
Results
Data collected using different restricted enzymes; Eco RI and Bam HI.

Act. BP
16,841 bp
12,275 bp
7,233 bp
6,527 bp
5,505 bp

Distance Travelled by Fragments Cut with HindIII

f(x) = - 0.01x + 1.89

Linear ()

Chart showing the line of best fit and equation used to find the base pairings of the
fragments cut by the restriction enzymes.
Image of the ideal gel used to collect data for graphing.

Procedure
1. Label four 1.5-mL tubes, in which you will perform restriction reactions: B for
BamHI, E for EcoRI, H for HinIII, and for no enzyme.
2. Use table for below as a checklist while adding reagents to each reaction. Read
down each column, adding the same reagent to all appropriate tubes; use a fresh
tip for each reagent. All groups share the same BamHI, EcoRI, HindIII enzymes at
a central station.
Tube
DNA
Buffer
BamHI
EcoRI
HindIII
B
4 l
5 l
1 l
E
4 l
5 l
1 l
H
4 l
5 l
1 l
4 l
5 l
3. Pool and mix reagents by tapping the tube on lab bench, or with a short pulse in a
microcentrifuge.
4. Incubate all reaction tubes for a minimum of 60 minutes at 37 degrees Celsius.
5. Retrieve pre-made gel
6. Add 1 l loading dye to each reaction tube. Mix dye with digested DNA by
tapping tube on lab bench, or with a microcentrifuge
7. Use micropipette to load contents of each reaction tube into a separate well in gel,
aligned as illustrated in Ideal Restriction Digest of Iambda DNA. Use a fresh tip
for each reaction tube.
a. Steady pipet over well using two hands
b. Be careful to expel any air in micropipette tip end before loading gel. (If
air bubble forms cap over well, DNA/loading dye will flow into buffer
around edges of the well)
c. Dip pipet tip through surface of buffer, position it over the well, and
slowly expel the mixture. Sucrose in the loading dye weighs down the
sample, causing it to sink to the bottom of the well.
8. Close top of electrophoresis chamber and connect electrical leads to an approved
power supply, anode to anode (red to red) and cathode to cathode (black to black).
Make sure both electrodes are connected to same channel of power supply.
9. Turn power supply on and set voltage as directed by your instructor. Shortly after
current is applied, loading dye can be seen moving through gel toward positive
pole of electrophoresis apparatus.
10. The loading gel will eventually resolve into two bands of color. The fastermoving, purplish band is the dye bromophenol blue; the slower-moving, aqua
band is xylene cyanol. Bromophenol blue migrates through gel at same rate as a
DNA fragment approximately 300 base pairs long. Xylene cyanol migrates as a
rate equivalent to approximately 2000 base pairs.
11. Allow the DNA to electrophoresis until the bromophenol blue band nears the end
of the gel.

H20
1 l

Gel Electrophoresis

Morgan Coulson
Honors Biology
May 25, 2016
Period 8

Materials

Agarose Gel
TBE Buffer Solution
Lambda DNA
Restriction Enzymes (EcoRI, BamHI, HindIII)
Micropipettes
Micropipette Tips
Hot Plate
Eppindorf Reaction Tubes
50 mL beakers
1000 mL flask
Electrophoresis Chamber
Graduated Cylinder
Microcentrifuge
Vortex
Ethidium Bromide Stain
Loading Dye
Gloves
Goggles
Staining Trays
Ultraviolet Light Source
Sharpie

Introduction
In the Gel Electrophoresis Lab we used restriction enzymes to cut DNA into
different fragments of DNA. A restriction enzyme is an enzyme that cuts the DNA
molecule in a specific location, the enzyme searching for a specific code or sequence,
called the recognition cite, to cut. These enzymes are most commonly used for DNA
cloning, inserting genes, criminal and paternity testing, gene technology,
etc.(learn.genetics.utah.ed). For criminal and paternity cases a special analysis is used
called, RFLP, or Restriction Fragment Length Polymorphism. We used the restriction
enzymes BamHI, EcoRI, and HindIII.
The process of Gel Electrophoresis is used to separate and analyze DNA and RNA
into smaller fragments, easier to obtain information from. Scientists use this process to
match the new small DNA fragments with the pieces from the original copies. DNA is
negatively charged because of all the phosphate groups in the backbone of the DNA.
Therefore, DNA is attracted to the positive electrode. As the pieces of DNA move
through the gel, they were feel resistance. Larger pieces of DNA will move at a slower
rate than smaller fragments of DNA(futurescienceleaders.org). RFLP electrophoresis,
the process used in paternity and criminal cases, is when a DNA sample is broken into
pieces and digested by restriction enzymes and the results are separated according to their
lengths by the process of gel electrophoresis. It was useful in finding out where a
specific gene disease lies on a chromosome. In addition it was also one of the first
methods used for genetic typing, or fingerprinting.(exploredna.co.uk).
The purpose of our gel electrophoresis lab was to see if the restriction enzymes;
BamHI. EcoRI, and HindIII, were able to cut DNA into different or the same sized
fragments of DNA. By doing the lab we were able to get good practice with restriction
enzymes and the process of gel electrophoresis. Lastly, we wanted to create a logarithmic
graph with the known data to figure out the other lengths of our DNA strands that were
created after the restriction enzymes cut them.
In the lab the independent variable(s) were the restriction enzymes which we used
BamHI, EcoRI, and HindIII. The dependent variable(s) were the sizes of the different
fragments, cut by the restriction enzymes. The control group would be the DNA without
any enzymes. We predict that the DNA will show different fragments and cuts, all
depending on what enzymes were used in the process of gel electrophoresis.

Discussion
Our hypothesis was that the restriction enzymes; EcoRI, BamHI, and HindIII,
would each cut the DNA differently, After completing the lab we found our hypothesis to
be correct. Each of the three restriction enzymes cut and moved through the gel
differently. We learned that the enzymes move differently because they look for different
codes and sequences. The larger fragments were the ones that traveled less of a distance
because they were larger and had a harder time moving. The smaller fragments moved
quicker because they were smaller and much easier to push through the gel. Some of the
sources of error could have been that we used an ideal picture of the gel rather than the
actual gel, because ours didnt work. This could have caused our measurements,
equations, and graphs to be off.

References
http://www.exploredna.co.uk/rflp-analysis.html
http://futurescienceleaders.org/researchers2012/2013/03/how-does-gel-electrophoresiswork/
http://learn.genetics.utah.edu/content/labs/gel/
The procedure was taken from Lab Manual from Carolina Biological Company