Gel Electrophoresis

Luc Madonna
Honors Biology
May 15, 2015
Period 5

Introduction
Restriction enzymes are enzymes that cut DNA in a certain way. These enzymes cut the
DNA in a certain pattern or code, which makes the DNA easier to comprehend. Scientists use
these restriction enzymes to identify a pattern in the DNA. They are essential in DNA
technology. Scientists use restriction enzymes for methods such as: gene technology, inserting
genes into a genome, and these can also be used on criminal investigations and paternity cases.
Gel electrophoresis is a scientific method of separating DNA, RNA, or proteins according
to molecular size. These molecules are pushed in an electrical field through a gel with small
wells. It is used to separate macromolecules, and in this case, DNA. (Biotechlearn.org) Since
DNA is negatively charged, it is attracted to the positive electrode. RFLP electrophoresis, or
restriction fragment length polymorphism, is a technique that exploits variations in homologous
DNA sequences (Wiki). In this, the DNA is broken into pieces by restriction enzymes and the
lengths are separated. It is an important tool in paternity and criminal cases. The purpose of the
lab was to see if different restriction enzymes cut DNA into different sized fragments or the same
sized fragments. Also, it was to practice with restriction enzymes and gel electrophoresis. Lastly,
it was to create a logarithmic graph with known data to figure out the lengths of the DNA
fragments that were created by different restriction enzymes. In this experiment, variables were
included. The dependent variable was distance traveled. This depended on the independent
variable, which were the three restriction enzymes. The control group was each strand of DNA.
The students hypothesis was that each distinct strand of DNA will have a different measurement,
based on each restriction enzyme used. Not much research was done prior to the experiment.
Materials
1. Agarose gel

2. Buffer Solution
3. Lamba DNA
4. Restriction Enzymes (EcoRI, BamHI, HindIII)
5. Micropipettes
6. Micropipette tips
7. Hot Plate
8. Eppendorf Reaction Tubes
9. 50 mL beakers
10. 1000 mL flasks
11. Electrophoresis Chamber
12. Graduated Cylinder
13. Micro centrifuge
14. Vortex
15. Ethidium Bromide stain
16. Loading Dye
17. Gloves
18. Goggles
19. Staining Trays
20. UV Light source

Procedure
Set Up Restriction Digest
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 restriction. 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
B
E
H
---

DNA
4 uL
4 uL
4 uL
4uL

Buffer
5 uL
5 uL
5 uL
5 uL

BamHI
1 uL
-------

EcoRI
--1 uL
-----

HindIII
----1 uL
---

H2O
------1

u
L

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.
***The teacher premade agarose gel***
Load Gel
1. Add 1 uL loading dye to each reaction tube. Mix dye with digested DNA by tapping
tube on lab bench, or with a pulse in microcentrifuge.

2. 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. (If air
bubbles forms cap over well, DNA/loading dye will flow into buffer around
edges of 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. Be careful not to punch tip of pipet
through bottom of gel.
Electrophorese
1. 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.
2. 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.
3. The loading dye will eventually resolve into two bands of color. The faster-moving,
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 at a rate equivalent to
approximately 2000 base pairs.
4. Allow the DNA to electrophorese until the bromophenol blue band nears the end of
the gel.
5. Turn off power supply, disconnect leads from the inputs, and remove top of
electrophoresis chamber.
6. Carefully remove casting tray and slide gel into staining tray labeled with your group
name. Take gel to your instructor for staining.

Ideal Gel from lab manual