Gel Electrophoresis Lab

Kelly Krolicki
Honors Biology
May 25, 2016
Period 9

Introduction:

Restriction enzymes are enzymes that cut DNA molecules into smaller fragments. The
cuts that are created by the restriction enzymes are made at a specific nucleotide sequence. As
the restriction enzymes vary, so do the DNA sequences. In labs, scientists use restriction
enzymes as a basic tool for several different processes including DNA cloning, DNA
fingerprinting, gene technology (inserting genes into a genome) and restriction fragment length
polymorphism, or RFLP, which is a technique that exploits variations in homologous DNA
sequences. Gel Electrophoresis is a method that scientists use to separate mixtures of DNA,
RNA, or proteins according to their molecular size. Gel Electrophoresis is used in everyday life
also. Scientists use it to get DNA fingerprints for forensic purposes, for paternity testing, to look
for evolutionary relationships among organisms, and even to check PCR reactions. The gel in
Gel Electrophoresis provides a resistance as molecules are pushed through it. Along with
separating molecules based on size, the gel also acts as an anti-convector by suppressing thermal
convection from the electrodes. Restriction Fragment Length Polymorphism also known as
RFLP, is a difference in homologous DNA sequences that is often detected by the presence of
fragments of different lengths after digestion of the DNA samples. The purpose of this lab was to
be able to see the different restriction enzymes cut DNA into different sized fragments or in some
cases, the same sized fragments. The lab allowed students to practice with and become more
comfortable with restriction enzymes as well as gel electrophoresis. With the results of this lab a
logarithmic graph is able to be created and the lengths of other DNA fragments that were created
by different restriction enzymes creating cutting them can be determined. The independent
variable of the experiment were the restriction enzymes, the dependent variable was the length of
the DNA fragments and the control group was the DNA without the restriction enzymes.
Hypothesis: Each restriction enzyme will cut the DNA fragments differently.
Materials:

Electrophoresis Chamber
Graduated Cylinder
Microcentrifuge
Vortex
Ethidium bromide stain
Loading Dye
Gloves
Goggles
Straining Trays
Ultraviolet Light Source
Sharpie

Agarose Gel
TBE Buffer solution
Lambda DNA
Restriction Enzymes (EcoRI, BamHI,
HindIII)
Micropipettes
Micropipettes tips
Hot Plate
Eppindorf Reaction Tubes
50 mL Beakers
1000 mL Flask

Procedure A: Set Up Restriction Digest

1. Label four 1.5 -mL tubes, in which you will preform 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 reagents 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

4 L

5 L

1 L

4 L

5 L

1L

4 L

5 L

1 L

4 L

5 L

1 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 37C. Your teacher may instruct
you to incubate the reactions for a longer period.
5. Add 1 L loading dye to each reaction tube. Mix dye with digested DNA by tapping tube on
lab bench, or with a pulse in microcentrifuge.
6. Incubate all reaction tubes for a minimum of 20 minutes at 37C. Your teacher may instruct
you to incubate the reactions for a longer period.
Procedure C: Loading Gel
1
Add 1 L 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 the Ideal Restriction Digest of Lamda DNA section. Use a fresh tip for
each reaction tube.
A. Steady pipette 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.

Procedure D: Electrophoresis
1. Close top of electrophoresis chamber and connect electrical to an approved power supply,
anode to anode (red-red) and cathode to cathode (black-black). Make sure both electrodes
are connected to 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 at a rate equivalent to approximately 2000 base pairs.
1.75
1.4

Logarithm of Actual Base Pair

1.05

f(x) = - 0.01x + 1.8

0.7

Linear ()

0.35
0
32.5

65

97.5

130

Distance (mm)
4. Allo
w the
DNA to electrophorese until the bromophenol blue band nears the end of the gel. Your
instructor may monitor the progress of electrophoresis in your absence; in that case; omit
steps 5 and 6.

5.

Turn off power supply, disconnect leads form 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.

Results:

Ideal gel taken from lab manual

Graph representing
the the log (kbp) on
the y axis and the
distance traveled in
mm on the x-axis.
Graph includes the
best fit line and a
linear equation.

HindIII
Dis.

EcoRI

Act. bp

Dis.

Cal. bp

Act. bp

BamH
I

Dis.

Cal. bp

Act. bp

42.0

*27, 491

42.0

21,066

21,226

47.0

17, 993

16,841

46.5

*23,130

63.0

10,861

7,421

52.0

15,369

12,275

60.5

9,416

71.0

8, 439

5,643

64.0

10, 524

7,233

70

6,557

77.0

6,984

4,878

66.0

9,881

6,527

83.9

4,361

91.0

4,491

3,530

72.0

8,177

5,505

115.5

2,322

123.0

2,027

**564

**125

Table including the distance traveled and the kbp length for each fragment. Including the EcoRI, BamHI,
HindIII, and the control

42

1.439

46.5

1.364

60.5

0.973

70

0.82

83.9

0.643

115.5

0.362

123

0.301

Chart holding the information used for the DNA restriction Analysis Graph.