Experiment 65 Restriction Endonuclease Cleavage of DNA

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

Obtain samples of selected restriction enzymes and reaction buffer (supplied with enzymes). Also obtain a DNA sample, either a viral (lambda) or bacterial plasmid. 

Using a micropipettor, add 10 pL of the reaction buffer to a small reaction vial or microcentrifuge tube. Add 15 pL of DNA. Then, with a fresh micropipet tip, add 15 pL of one enzyme. 

Repeat step 2 using separate vials for each additional enzyme. 

Mix all tubes by microcentrifuging for 5 min at 15,000 rpm, or by tapping tubes on any flat surface at least 50 times. Then incubate in a water bath at 37°C for 50 min. Carry these tubes over to Experiment 66. 

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