Digesting DNA Using Restriction Enzymes

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Presentation transcript:

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).

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 samples 2. Sterile Water 3. Restriction Enzyme 4. Buffer 5. 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.

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.