Gel Electrophoresis Lab

Colton Eisenreich
Biology Period
05/25/16
Introduction
Gel Electrophoresis is a method used to separate DNA according to size. In the process
restriction enzymes cut the DNA at certain points. They cut at places called palin-drones these
are spots where the DNA is read the same way forwards and backwards. The now cut DNA is
placed in wells in a gel. This gel is put into a chamber that is then charged with positive and
negative electricity. DNA is positively charged so it moves towards the negative and of the
chamber. The DNA has to push its way through the gel and the size of the DNA pieces
determines how far it will go. Since all DNA is different it will be cut in different places. This
makes the pieces bigger or smaller and they will travel a different distance. This process is called
RFLP (Restriction Fragment Length Polymorphism). This is used to test DNA in a crime scene
to try to match with suspects DNA. The purpose of this lab was to see how different restriction
enzymes effected the same DNA and to practice using restriction enzymes and gel
electrophoresis. Then to create a logarithmic graph with known data to figure out the length of
the other fragments. The independent variable is the restriction enzyme used and the dependent
variable is the distance traveled by the fragments. The control group was DNA with no
restriction enzyme added. The scientist hypothesizes that the different enzymes will cause DNA
to form large streaks that will travel different distances.

Materials
• Agarose gel
• TBE Buffer solution
• Lambda DNA
• Restriction Enzymes (EcoRI, BamHI, HindIII)
• Micropipette tips
• Micropipettes
• 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 kliht source
• Sharpie
Procedure
1. Label four 1.5-mL tubes, in which you will perform restriction reactions: B for BamHI, E for
EcoRI, H for HindIII, and – for no enzyme.
2. Use table 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 H2O
B 4 µL 5 µL 1 µL - - -
E 4 µL 5 µL 1 µL - -
H 4 µL 5 µL - - 1 µL -
- 4 µL 5 µL - - - 1 µL

3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a short pulse in a
microcentrifuge.
4. Incubate all reaction tubes for a minimum of 20 minutes at 37* C. Your teacher may instruct
you to incubate the reactions for a longer period.
5. The teacher proceded to create the gels in order to save time for the lab
6. Add 1 µL loading dye to each reaction tube. Mix dye with digested DNA by tapping tube on
lab bench or with a pulse 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 Lambda 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.
c. Dip pipet through surface of the buffer, position it over the well and slowly expel the mixture.
Sucrose in the loading dye weighs down the sample, sinking it to the bottom of the well. Do not
punch tip through the bottom of the well.
8. Close top of electrophoresis chamber and connect electrical leads to an approved power
supply, anode to anode (red-red) and cathode to cathode (black-black). Make sure both
electrodes are connected to the same channel of power supply.
9. Turn power supply on and set voltage as directed by instructor.
10. The loading dye will eventually resolve into two bands of color. The faster-moving purple
band is the dye, bromophenol blue; the slower moving aqua band is Xylene cyanol.
Bromophenol blue migrates through the gel at the same rate as DNA with 300 base pairs and the
xylene cyanol moves at a rate equivalent to the rate of DNA with approximately 2,000 base
pairs.
11. Allow the DNA to electrophorese until the bromothymol blue band is near the end of the gel.
12. Turn off the power supply, disconnect the leads, and remove the lid to the electrophoresis
chamber.
13. Carefully remove casting tray and slide gel into staining tray. Take staining tray to instructor
for staining.

Results

Distance Traveled by Base Pairs

1.6
1.4
1.2
1
log(kbp)

0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120 140
Distance(mm)

This graph shows the distance travel by the log of the Kbase pairs. The base pairs were given to
the scientist.
HindIII
Distance(mm) logKBP
42 1.439
46.5 1.3636
60.5 0.973
70 0.8195
83.5 0.63346
115.5 0.361727
123 0.30103
This is the table used for the graph with the distance traveled and the log of the given Kbase
pairs.
Analysis
The scientist’s hypothesis was not correct. The enzyme cut the DNA into different
lengths. Each group of base pairs the same length traveled a certain distance according to their
length. The smaller fragments traveled further than the larger fragments that couldn’t travel as
far through the gel. Error could have come from many places. It could have come from how
much DNA we used. There may have also been an error in shipping of the DNA causing damage
to it. There could have also been human errors in calculation.
 Source
http://www.nature.com/scitable/definition/gel-electrophoresis-286
https://askabiologist.asu.edu/restriction-enzymes
http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
Carolina Biology supply company lab manual’s