DNA Restriction Analysis

Justin Koenig
Honors Biology
Period 9
25 May 2016

Introduction:
Specific DNA sequences are recognized and bound to Restriction enzymes. The enzymes cleave
the DNA within the sequence. After, they (the enzymes) enter the bacteria and cut the viral DNA into
multiple fragments. Restriction enzymes are used to isolate specific genes and regions of the genome.
When the DNA is cleaved, it creates fragments that are unique to every person. DNA can be manipulated
by inserting genes into genomes. Restriction enzymes are a crucial way to solve criminal and paternity
cases because gene information is extracted during the process. Polymorphism determines each of the
DNA fragments length. The lengths of the fragments are compared. If they match up, it proves who the
criminal or father is. In order to group these DNA fragments, scientists must use gel electrophoresis.
When an electric current is applied, DNA fragments move toward the positive end of the gel. Smaller
fragments move faster and farther than larger ones. The grouping creates a pattern that is matched with
other patterns. The lab was conducted to determine if different restriction enzymes would cut DNA into
different or same sized fragments. Students learned how to use with restriction enzymes and gel
electrophoresis. Students also created a logarithmic graph in order to figure out the lengths of DNA
fragments that were cut by restriction enzymes. The independent variable is the addition of different
restriction enzymes. The dependent variable is the size of the fragment. I hypothesize that 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
5. 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
6. 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
7. Steady pipet over well using two hands
8. 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.)
9. 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.
10. 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
11. 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
12. 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
13. Turn off water supply, disco
Distance Travelled by Fragments Cut with HindIII
130
f(x) = - 69.11x + 135.61
97.5

Fragment Size (log kbp)

65

Linear ()

32.5

0
0

0.4

0.8

Distance travelled by fragment (mm)

nnect leads from the inputs, and remove top of electrophoresis chamber
14. Carefully remove casting tray and slide gel into staining tray labeled with your group name. Take gel to
your instructor for staining

This picture is gel electrophoresis used in the

experiment.

1.2

Results:
Ideal Restriction Digest of Lambda DNA
HindIII

EcoRI

BamHI

Dis. (cm) Act. bp

Dis. (cm) Cal. 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

Act. Bp

Control

Dis. (cm) Cal. bp

Act. bp

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. The enzymes split the DNA at different parts. The smaller fragments moved
further through the gel whereas larger fragments moved less. The calculated bp and actual bp were
different because the calculated is just a prediction.
References:
Biology Book
https://askabiologist.asu.edu/restriction-enzymes