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Gel Electrophoresis

John Fukon
Honors Biology
5/15/16
Period 5

Introduction:
Restriction enzymes are enzymes that are used to chop up DNA. They cut both nucleotide
strands, breaking the DNA into fragments. Scientists use restriction enzymes to cut DNA
into smaller fragments. The cuts made by the enzyme are always at a specific DNA
sequence. Gene Technology has increased for genes are being inserted into a genome to
allow for cures of diseases and other new possibilities. RFLP, or Restriction Length
Polymorphisms, are used in criminal cases to match the DNA of the suspects to the
victim. In this process, scientists can find out whether or not a suspect is guilty based off
DNA evidence. Gel electrophoresis is the separation of nucleic acids and proteins
according to their size and charge. It is used to separate DNA according to its size and
give scientists a DNA fingerprint. Gel electrophoresis works by first loading the samples
onto the gel and having an electric current applied to the gel. Since DNA is negatively
charged, the current forces the samples of DNA to move towards the opposite end of the
gel. The smaller pieces will move faster than the larger ones. This is because the small
ones can easily fit through the gel whereas the larger ones cannot fit as easily, therefore,
slower. RFLP gel electrophoresis is resulting restriction fragments that are separated due
to their lengths. The DNA sample pieces are in their location for measurements and for
analysis on similarities between two individuals. This is very helpful in criminal and
paternity cases for it shows the similarities between two people. For example, if there are
multiple suspects thought to have killed someone, scientists can test their DNA and get
these results. Based off these results, a suspect can be found guilty or innocent. If the
DNA matches, then they are similar and can be found guilty. The purpose of this lab was
to see if different restriction enzymes cut DNA into different sized fragments or the same

sized fragments. During this lab, the class wanted to practice with restriction enzymes
and gel electrophoresis, and the class wanted 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 independent variable in this lab is the size of the
DNA fragment after being cut by the Restriction Enzyme. The dependent variable in this
lab is the distance that the DNA fragment traveled. The control group is the uncut DNA
that was not treated with the restriction enzyme but still run through the entire process.
The hypothesis of this lab is that each restriction enzyme will cut the DNA in different
places which causes different sizes of DNA fragments. Due to this, the smaller DNA
fragments will travel farther than the larger DNA fragments.
Materials:
1. Agarose Gel
2. TBE Buffer solution
3. Lambda DNA
4. Restriction Enzymes (EcoR1, BamHI, HindIII)
5. Micropipettes
6. Micropipette tips
7. Hot plate
8. Eppindorf reaction tubes
9. 50 mL beakers
10. 1000 mL flask
11. Electrophoresis chamber
12. Graduated cylinder
13. Microcentrifuge
14. Vortex
15. Ethidium bromide stain
16. Loading dye
17. Gloves
18. Goggles
19. Staining trays
20. Ultraviolet light source
21. Sharpie
Procedure: 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 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 badn is xylene
cyanol. Bromophenol blue migrates through the 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. 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 labeled with your group name.
Take gel to your instructor for staining.
Results:

Distance Traveled by Fragments Cut with HindIII


1.6
1.4
f(x) = - 0.01x + 1.9

1.2
1

Fragment Ssize (log kbp) 0.8


0.6
0.4
0.2
0
30 40 50 60 70 80 90 100110120130

Distance Travelled by Fragment (mm)

HindIII
Dis.
(mm)
42.0
46.5
60.5
70.0
83.9
115.5
123.0

Act. bp
27,491
23,130
9,416
6,557
4,361
2,322
2,027

EcoRI
Dis.
(mm)
42.0
63.0
71.0
77.0
91.0

Cal. bp
21,066
10,861
8,439
6,984
4,491

BamHI

Act. bp

Dis.

Cal. Bp

Act. Bp

21,226
7,421
5,643
4,878
3,530

(mm)
47.0
52.0
64.0
66.0
72.0

17,993
15,367
10,524
9,881
8,177

16,841
12,275
7,233
6,527
5,505

Discussion:
The hypothesis was proved to be true in the ideal gel that was used in the lab. However, the
actual gel used by the class was not