Overview:

This worksheet teaches you how to use Benchling to choose restriction enzymes to make DNA digests, and evaluate the results with gel electrophoresis. Best of all, it doesn’t require any experimental setup and can be done entirely in silico.

Content and materials for this module were co-developed with Dr. Philip Leftwich, Biology Lecturer at the University of East Anglia (Norwich, UK)

Prior knowledge:

A basic understanding of the concept of restriction enzymes and site-specific DNA cutting, and gel electrophoresis to separate DNA fragments by size. Check out these resources for background on the theory of restriction enzymes and gel electrophoresis.

Learning outcomes:

By the end of this worksheet you will be able to:

  • Choose restriction enzymes to cut DNA
  • Make predictions on DNA fragment sizes from restriction digests
  • Integrate gel images and virtual digests into lab notebooks

Before you begin:

Learn how to get started and sign up with Benchling here. Start this worksheet once you have an account so you can create your own copy of these files to edit. Sequences in this worksheet can be copied directly into your Projects while notebook entries will require manual copy & paste into a new blank entry.


Worked example:

For any DNA sequence such as a plasmid or PCR product, Benchling can recognize cut sites for restriction enzymes and simulate the expected fragments of DNA after digestion with them. Different digests can be displayed on a virtual gel for students to predict the expected band patterning. Follow along with the exercise in this short video below to produce new DNA fragments after digestion of a vector with different restriction enzymes:

  • Copy the pBAD Vector into your own Project. Open and switch the view to “Linear Map”.
  • Open the “Digests” button, navigate to “New Digest”, and specify “NEB” and “Double Cutters” in the settings.
  • Search by the number of cuts, “1”, find and left-click on “AseI” to select this enzyme, and hit “Run Digest”.
  • Save the digest as “Linearized” and navigate to the “Virtual Digest” tab to see the simulated gel image.

Question(s):

What’s the difference between a single and double cutter when choosing restriction enzymes?

Answer this question on your own and check the "Solution" at the bottom.

Practice:

Can you repeat the exercise above, replacing AseI for different enzymes?

Add digests with restriction enzymes with ClaI - HincII (Pair 1) and AccI - BglII (Pair 2) and then view the *Virtual Digest* again.

  • Repeat steps 1 & 2, at step 3 choose two enzymes ClaI & HincII
  • Repeat step 4, saving this digest as Pair 1
  • Repeat this process one more time for Pair 2, AccI-BglII (You should now have three virtual digests)
  • Analyze the size of each band by clicking on it and specify a commercial DNA ladder or create your own.
  • Change the order of the lanes on the Virtual Digest tab by rearranging the digest tabs.

Stretch yourself:

Check out how to perform an actual gel electrophoresis run in the lab using this example entry. Use screen-capture to take a snapshot of your virtual gel and paste it as an image into a new Notebook entry. Write out a protocol for how you would carry this out in the lab.

Benchling gives you the ability to compare any digests you perform with a simulated gel from the Virtual Digest function allowing you to see if the results matched their predictions.

Solution(s):

This has nothing to do with the properties of the restriction enzyme but specifies how many target cut sites are available for that enzyme on this DNA template. An enzyme might have 0, 1, 2, or many cut sites, and this will change depending on the DNA sequence.

Congrats! You've finished the learning module: Gel Electrophoresis and Restriction Enzymes.

What’s next?

Check out this next module and teach your students all about: Molecular Cloning Methods.

Or learn more about Benchling for Education.

Did this answer your question?