DNA Detectives Part C: Preparation of DNA Samples
Objectives:
 Assume the role of a forensic scientist in an attempt to solve a violent crime using DNA evidence
 Perform the process of restriction enzyme digestion of DNA
 Employ agarose electrophoresis to separate the digested DNA fragments
 Identify the guilty suspect based on the DNA fingerprinting evidence
 Understand the principles and practices of DNA technology as applied to the forensic sciences
Materials Needed:
0.8% agarose gel on gel tray
TBE running buffer 1X, 350 ml (approximately)
Quikview DNA Stain
Loading Dye
Gel Staining tray
Microcentrifuge tubes
Micropipets
Electrophoresis Chamber
Power Supply
Incubator or hot water bath
DNA Samples:
Crime Scene DNA
Suspect #1 DNA
Suspect #2 DNA
Suspect #3 DNA
Suspect #4 DNA
Lambda DNA/Hind III marker
Pvu II restriction enzyme,
Pvu II reaction buffer 10X
Scenario
Police were called to the scene of a homicide. Found at the scene was a large amount of blood. Blood
typing revealed that not only was the blood of the victim present at the scene, but also blood from another
person, assumed to be the guilty party. Blood evidence was collected at the scene. Blood samples from
four suspects were also drawn under court order and all samples were found to be of the same blood type as
the blood believed to have come from the perpetrator of the crime. The police have turned to you and your
fellow lab technicians to employ the process of DNA fingerprinting in building a case against one of the
suspects.
Procedures: Part I Restriction Enzyme Digestion of DNA
1.
2.
With a permanent marker, label five microcentrifuge tubes as follows: Crime Scene; Suspect 1;
Suspect 2; Suspect 3; and Suspect 4
Using a fresh micropipette tip each time, add the exact amounts of the following reagents, (note:
when dispensing small volumes, it may be helpful to place the pitet tip against the side of the
microcentrifuge tube and then expel the liquid) in the order listed, to each microcentrifuge tube:
3.

Crime Scene:

Suspect #1

Suspect #2

Suspect #3

Suspect #4
Crime Scene DNA
Pvu II Reaction Buffer 10X
Pvu II Restriction Enzyme
Suspect #1 DNA
Pvu II Reaction Buffer 10X
Puv II Restriction Enzyme
Suspect #2 DNA
Pvu II Reaction Buffer 10X
Puv II Restriction Enzyme
Suspect #3 DNA
Pvu II Reaction Buffer 10X
Puv II Restriction Enzyme
Suspect #4 DNA
Pvu II Reaction Buffer 10X
Puv II Restriction Enzyme
10 µl
2 µl
2 µl
10 µl
2 µl
2 µl
10 µl
2 µl
2 µl
10 µl
2 µl
2 µl
10 µl
2 µl
2 µl
4.
5.
6.
Spin each tube down in the microcentrifuge for approximately 30 seconds to mix all of the
reagents.
Incubate all five tubes at 37º C for one hour using a foam flotation device.
Add 2 µl of loading dye to each tube. To stop the enzyme activity in the enzyme reaction tubes,
incubate all five tubes in the foam flotation device for five minutes at 65° C. This will denature the
enzyme.
Procedures: Part II Loading and Running a Gel
1.
2.
3.
4.
5.
6.
7.
8.
Reflect on the dry or submarine methods, and choose which one you would rather employ.
Before loading, make sure your wells are nearest to the NEGATIVE (black) end of the chamber!
Using either the dry or submarine method, load each DNA sample into the corresponding gel well
listed below:
Lane #1: Lambda DNA/Hind III marker, 10 µl
Lane #2: Crime Scene DNA, 15 µl
Lane #3: Suspect #1 DNA, 15 µl
Lane #4: Suspect #2 DNA, 15 µl
Lane #5: Suspect #3 DNA, 15 µl
Lane #6: Suspect #4 DNA, 15 µl
After you’ve loaded your gels, attach the cover, and cords to the power supply. Check to make
sure entire table surface is dry. Then call teacher over to inspect. Once teacher gives you the goahead, you may plug in your units, and begin to electrophorese. Run your gel on 125V in hopes of
having DNA travel as far as possible in the coming hour.
Observe bubbles from electrodes to make sure unit is working.
Turn off power supply when the loading dye has run half-way off the end of the gel. Unplug the
power supply. Wait 10 seconds, and then open the unit carefully. Remove the gel from the casting
tray in the chamber.
With time permitting, stain and destain the gels, and make observations on the transilluminator
(light box).
Or…store your gels in baggie with some buffer solution overnight. Stain and destain tomorrow.
Wash hands thoroughly.