DNA Restriction Analysis

Brooke Horton
Honors Biology
Period 9
25 May 2016

Introduction:
Restriction enzymes recognize and bind to specific DNA sequences and cleave the DNA within
that sequence. The enzymes cut the viral DNA into fragments after it enters the bacteria. Scientists use
restriction enzymes for isolating specific genes or regions of the genome. When the restriction enzymes
cleaves genomic DNA, it creates fragments of different sizes that are unique to every person. Scientists
insert genes into genomes, total DNA present in the nucleus of a cell, with DNA tools that can also be
used to manipulate DNA. These tools are also useful when solving criminal and paternity cases because
of the gene information extracted from this process. Restriction fragment length polymorphism is used
to determine the length of each DNA fragment. (Biology book) Criminal and paternity cases compare
the lengths of the fragments, and if they match up, scientists can prove which individual it was extracted
from. To group these DNA fragments, scientists use gel electrophoresis. This is when an electric current
is applied, and DNA fragments move toward the positive end of the gel. The smaller fragments move
faster and farther than the larger fragments. This grouping creates a particular pattern that can be
compared to other patterns. Scientists can also compare these patterns to solve criminal and paternity
cases. RFLP gel electrophoresis splits the fragments and groups them to create patterns compared in
criminal and paternity cases. This lab was conducted to see if different restriction enzymes cut DNA into
different sized fragments or the same sized fragments. This also gave students good practice with
restriction enzymes and gel electrophoresis. The students also created a logarithmic graph with known
data to figure out the other lengths of the DNA fragments that were created by different restriction
enzymes cutting them. The independent variable is the addition of different restriction enzymes. The
dependent variable is the size of the fragment. I hypothesis that the DNA will be split differently. The
controlled group of the DNA did not have an enzyme, so it was not split.
Materials:
Agarose Gel
TBE Buffer solution
Lambda DNA
Restriction Enzymes (ECOR1, BamHI, HindIII)
Micropipette tips
Hot plate
Eppindorf reaction tubes
50 mL beakers
1,000 mL flask
Electrophoresis chamber
Graduated cylinder
Microcentrifuge
Vortex
Ethidium Bromide Stain
Loading dye
Gloves
Goggles
Staining trays
Ultraviolet light source
Sharpie
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 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
(uL)

Buffer
(uL)

BamHI
(uL)

EcoRI
(uL)

HindII
(uL)

Water
(uL)

3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a short pulse in a micro
centrifuge
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.
Load Gel
1. A pre-made gel was retrieved and used
2. Add 1 uL loading dye to each reaction tube. Mix dye with digested DNA by tapping tube on lab
bench or with a pule in micro centrifuge
3. Use micro pipet to lead 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
4. Steady pipet over well using two hands
5. Be careful to expel any air in micro pipet tip end before leading gel. (If air bubble forms cap over
well, DNA/loading dye will flow into buffer around edges of well.)
6. Dip pipet tip through surface of buffer, position it over the well, and slowly expel the mixture. Surges
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
(red-red) and 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 through gel at same rate as a DNA fragment approximately 300 base pairs long. Xylene
cyan 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 water 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

Results:
This a picture of the ideal gel electrophoresis
used in the experiment.

Distance Travelled by Fragments Cut with HindIII

Fragment Size (log kbp)

130

y = -69.107x + 135.61

97.5

65

32.5

0
0

0.4

0.8

1.2

Distance travelled by fragment (mm)

1.6

HindIII

EcoRI

Dis.
(cm)

Act. bp

BamHI

Dis.
(cm)

Cal. bp

Act. bp

Dis.
(cm)

Control
Cal. bp

Act. bp

27,491

22,440

24,756

4.9

16,890

16,841

4.5

23,130

4.6

18,570

21,226

6.7

9,570

12,275

5.9

9,416

6.6

9,880

7,421

6.9

8,990

7,233

6.9

6,557

7.4

7,680

5,643

7.5

7,440

6,527

8.2

4,361

7.9

6,560

4,878

11.4

2,322

9.3

3,318

3,530

12.2

2,027

Dis.
(cm)

Act. bp
4.3

5,505

(this is the result table for the distance and bp length for each fragment)
Discussion:
My hypothesis was correct because the enzymes split the DNA at different parts. The smaller the
fragment, the further it was throughout the gel. There was a difference between the calculated bp and the
actual bp because the trend line may have been off, or I could have measured incorrectly.
References:
Biology Book
https://askabiologist.asu.edu/restriction-enzymes
http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechRFLP.shtml