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Gel Electrophoresis
Drew Linarelli
Honors Biology
17 May 2016
Period 9

Introduction

A restriction enzyme is an enzyme that cuts DNA at or near a specific point

known as recognition sites. They mainly work by shape to shape matching. When
the enzyme meets an area of the DNA that has the same sequence of the enzyme,
the enzyme causes a break in both strands of the DNA molecule. Scientists mainly
use them to cut DNA into smaller fragments which could help a scientist in many
ways. For instance in Genetic engineering, restriction enzymes are used to cut the
DNA into fragments. They are also used to create recombinant DNA, which is when
scientists bring together genetic material, such as restriction enzymes, from
multiple sources creating sequences that would not otherwise be found in a
genome. Restriction enzymes are also used in RFLP analysis, which is when a DNA
sample is broken into fragments and then separated according to their lengths by
gel electrophoresis. Through this scientists are now able to find similarities in DNA,
which is very helpful for solving criminal and paternity cases.

Gel electrophoresis stated by google, Is a laboratory method used to

separate mixtures of DNA, RNA, or proteins according to molecular size. In gel
electrophoresis, the molecules to be separated are pushed by an electrical field
through a gel that contains small pores. Gel electrophoresis is used to separate
macromolecules like DNA and RNA, according to their size. It is also used to
separate proteins according to their size and their charge. Furthermore RFLP gel
electrophoresis is when electrophoresis gel separates the restriction fragments
according to their size in this analysis. This method can be used for criminal and
paternity cases to differentiate and identify the similarities based on the lengths
concluded by the gel of the DNA strand to identify possibly the same strand of DNA
or even the exact opposite strand, which could solve these types of cases.

First the purpose of this lab is we want to see if different restriction enzymes
cut DNA into different sized fragments or the same sized fragments. Second we
want to get practice with using tools and materials such as restriction enzymes and
gel electrophoresis. Finally we want to create a logarithmic graph with known data
to figure out the other lengths of our DNA fragments that were created by different
restriction enzymes cutting them.

The dependent variable is the size of the fragments cut by the restriction
enzymes while the independent variable is the different restriction enzymes. Then
the control group is the DNA that does not consist of any restriction enzymes. The
hypothesis is that the restriction enzymes will cut the DNA in different places, the
smaller fragments will move faster through the gel, and the control group will not
move very much if at all.

Materials List

Agarose Gel
TBE Buffer Solution
Lambda DNA
Restriction Enzymes (EcoRI, BamHI, HindIII)
Micropipettes
Micropipettes tips
Hot plate
Eddindorf reaction tubes
50 mL beakers
1000 mL flask
Electrophoresis cylinder
Microcentrifuge
Vortex
Ethidium bromide stain
Loading dye
Gloves
Goggles
Staining trays
Ultraviolet light source
Sharpie

Procedures
Procedure A: Set up Restriction Digest
1.
Label four 1.5-mL tubes, in which you will perform restrictions: 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 the appropriate tubes; use a
fresh tip for each reagent. All groups share the same BamHi, EcoRI, HindIII enzymes
at a central station
3.
Pool and mix ingredients by tapping the tube bottom on the lab bench, or
with a short pulse in a microcentrifuge

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4.
Incubate all reaction tubes for a minimum of 20 minutes at 37 degrees
Celsius. Your teacher may instruct you to incubate the reactions for a longer period.
Procedure B: Load Gel
1.
Add 1 mean of 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 separated well
in gel, aligned as illustrated in ideal Restriction Digest of Lambda DNA.
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 form 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 C: 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 (blackblack). 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 electrophorese apparatus.
3.
The loading dye 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 at a rate
equivalent to approximately 2000 base pairs.
4.
Allow 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 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 your get to your instructor for staining.

Results
Gel Electrophoresis lab

This is the gel on a light box that I examined to find all of my measurements and
results for this lab.

Ideal Restriction Digest of Lambda DNA (HindIII)

1.6

1.4
f(x) = - 0.01x + 1.9
1.2

Fragments size (log kbp)

0.8

0.6

0.4

0.2

0
30

40

50

60

70

80

90

100

Distance travelled by fragmets (mm)

This graph shows the results of the HindIII data generating a best fit line. I
also used the equation at the top to find the base power of my other two
variables.

110

120

This chart shows the distance and the actual base pairs for HindIII. The
information in the middle of the chart was also used to create the graph on
the previous page. The info is the log of kbp. Distance is in mm.

This chart shows the distance, the calculated base pairs, and the actual base
pairs for EcoRI. Distance is in mm.

This chart shows the distance, the calculated base pairs, and the actual base
pairs for BamHI. Distance is in mm.

This chart shows the distance and calculated base pairs of an area with no
restriction enzymes. Which is the control group in this lab. Distance is in mm.

Analysis
The hypothesis was that the restriction enzymes will cut the DNA in different
places, the smaller fragments will move faster through the gel, and the control
group will not move very much if at all. Therefore my hypothesis was absolutely
correct. I was shown that the restriction enzymes really do cut the DNA in different
locations. I also found out that the smaller fragments move the farthest containing
the least amount of base pairs, especially shown in HindIII of the Gel
Electrophoresis picture on page five. However I had some sources of error while
doing the lab. We did not actually use real gel to measure these results because the
DNA fragments did not show up in my gel due to an unknown problem, so all we
used was a picture, which is on page five. Therefore is it was hard to measure the
exact points on the picture, which is the reason why some of my calculated base
pairs are not incredibly accurate to the actual base pairs.

References
(Scienceclarified) Genetic Engineering using restriction enzymes source,
http://www.scienceclarified.com/scitech/Genetics/Genetic-Engineering.html

(google) Google used in-text citation for how Gel electrophoresis works,
https://www.google.com/?gws_rd=ssl#q=gel+electrophoresis

(biology-pages) Information on restriction enzymes source,

http://www.biology-pages.info/R/RestrictionEnzymes.html

(biotechlearn) Gel electrophoresis information source,

http://biotechlearn.org.nz/themes/dna_lab/gel_electrophoresis

(kau.edu) Mutations done by restriction enzymes source,

http://www.kau.edu.sa/Files/0002923/Files/18591_Restriction%20Fragment
%20Length%20Polymorphism.pdfa

Lab manual procedures source,

Carolina Biological supply company, 2700 York Road, Burlington, North
Carolina 27215, DNA Restriction Analysis