DNA Structure and Analysis

Activity 4.1: Restriction Site Prediction Using NEBcutter

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Activity 4.1: Restriction Site Prediction Using NEBcutter

Research Questions:– How many times does a given restriction enzyme cut lambda

DNA?– When cutting lambda DNA with a given restriction enzyme,

what pattern will the resulting DNA fragments make on a gel? Objectives:

– Use NEBcutter to predict where restriction enzymes will cut the bacteriophage lambda DNA sequence

– Produce a virtual gel image of lambda DNA cut with a specific restriction enzyme

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Skills to Master

Use NEBcutter software to predict restriction enzyme sites

Record detailed software instructions into a laboratory notebook

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Student Workstation Materials

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Protocol Highlights/Tips

Go to the NEB website– tools.neb.com/NEBcutter2/

Select Lambda under Standard sequences

Ensure that the radio button for Linear sequence is selected

Write detailed steps into notebook

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Summary

Make sure to:– Record all steps in your notebook– Lambda sequence is selected under standard

sequences– Sequence is selected as linear– Print the fragments list and gel image to paste

into your notebook– Adjust the gel type to 1% agarose and adjust the

marker to Lambda-HindIII Digest– DNA type should be unmethylated