Digesting DNA Using Restriction Enzymes

Purpose

• To learn the principles of DNA fingerprinting.

• To understand endonuclease activity for genetic engineering and biotechnology.

Introduction

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

EcoR1 G A A T T C C T T A A G G A A T T C C T T A A G

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.

Introduction

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

Materials

• Latex Gloves• Safety Glasses• DNA• Restriction Enzyme• Buffer• BSA• Microcentrifuge

tubes

• Microcentrifuge Rack

• 0.5-10 uL Micropipetter

• 20-200 uL Micropipetter

• Micropipette Tips• Permanent Marker

Methods

• Restriction Enzyme Assay• Gel Electrophoresis

Getting Started

• Gloves and safety glasses should be donned throughout this assay.

• This helps to prevent contamination of the DNA.

Getting Started

• Set up a microcentrifuge rack as follows:

1. DNA samples2. Sterile Water3. Restriction Enzyme4. Buffer5. BSA

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.

Procedure

• Use the 20-200 uL micropipetter to transfer 26 uL sterile water into each reaction tube.

– The same tip may be used throughout this step.

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.– The same tip may be used throughout this step.

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.

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.

Procedure

• Mix the reaction cocktail by gently tapping the sides of the tubes.

• Do not:– Shake– Invert – microfuge

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.