Download - Digesting DNA Using Restriction Enzymes
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.