1. Digesting DNA Using Restriction Enzymes

2. Purpose To learn the principles of DNA fingerprinting.
To understand endonuclease activity for genetic engineering and biotechnology.

3. Introduction
Restriction endonucleases (restriction enzymes) are capable of cutting both strands of DNA at one specific site in the nucleotide sequence (recognition site).

4. Introduction Where the DNA is cut depends on its base sequence.
Unrelated strands of DNA will be consequently cleaved into various smaller size pieces.
One DNA strand may have 5 pieces while another may have 12 pieces.

5. Introduction
These DNA fragments can then be analyzed by gel electrophoresis for pattern comparison known as DNA fingerprinting.

6. Materials Latex Gloves Safety Glasses DNA Restriction Enzyme Buffer
BSA
Microcentrifuge tubes
Microcentrifuge Rack
uL Micropipetter
uL Micropipetter
Micropipette Tips
Permanent Marker

7. Methods
Restriction Enzyme Assay
Gel Electrophoresis

8. Getting Started
Gloves and safety glasses should be donned throughout this assay.
This helps to prevent contamination of the DNA.

9. Getting Started Set up a microcentrifuge rack as follows:
1. DNA samples
2. Sterile Water
3. Restriction Enzyme
4. Buffer
5. BSA

10. Getting Started Label 5 new microcentrifuge tubes as follows:
Suspect A
Suspect B
Suspect C
Suspect D
Crime Scene (CS)
These are the reaction tubes.

11. Procedure
Use the uL micropipetter to transfer 26 uL sterile water into each reaction tube.
The same tip may be used throughout this step.

12. Procedure Use the 0.5-10 uL micropipetter to transfer
4 uL buffer into each reaction tube.
The same tip may be used throughout this step.
Use the same technique to transfer 4 uL BSA into each reaction tube.

13. Procedure
Use the 0.5 uL micropipetter to transfer 1 uL of stock DNA into its corresponding reaction tube.
Important: Tips should be switched out between each tube to prevent cross contamination of DNA samples.

14. Procedure
Use the 0.5 uL micropipetter to transfer 5 uL restriction enzyme into each reaction tube
Be sure to change tips between each tube.

15. Procedure
Mix the reaction cocktail by gently tapping the sides of the tubes.
Do not:
Shake
Invert
microfuge

16. Procedure Incubate the tubes at room temperature overnight.
After incubation the samples are ready to be analyzed by gel electrophoresis.
Samples can be stored frozen if needed.