Prior knowledge:

A basic understanding of the concept of DNA cloning and familiarity with related concepts such as PCR, restriction enzymes, and DNA synthesis.


See previous worksheets on Benchling that introduce PCR and restriction enzyme digests.

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

Learning outcomes:

  • Plan and design molecular cloning strategies
  • Compare different cloning methods including restriction, Gibson and Golden Gate cloning
  • Integrate plasmid maps 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:

We’ll first demonstrate how to do traditional restriction cloning (digest-and-ligate) using Benchling.

Type II restriction enzymes are commonly used in restriction cloning workflows. These enzymes cleave DNA sequences into fragments at or near specific recognition sites. The cleavage can produce “sticky” or “blunt” ends of DNA that can be ligated to other DNA fragments that possess compatible ends. We’ll look at an example of restriction cloning below. The fictional JfyP gene is inserted into the pET 31b plasmid using ClaI and AlwNI restriction sites.

Digest and Ligate Cloning - Assembly Wizard
Follow the instructions below to use the Benchling Assembly Wizard to plan and design cloning for your DNA constructs.

  • Open the “Assembly Wizard” and choose “Digest and Ligate” as your assembly method.
  • Work on setting your backbone first and using the pET-31b sequence. Holding down “Ctrl+Shift” (or “Cmd + Shift” for Mac) and click on both the ClaI and AlwNI restriction sites to highlight the area between both restriction sites.
  • Now “right-click” to open a new menu and choose “Invert Selection” to highlight this area and choose “Set from Selection” to finalize your backbone.
  • Choose the insert in your assembly and switch to the JfyP Insert sequence. Hold down “Ctrl+Shift” to click on the ClaI and AlwNI sites again.
  • If you get a “green check” and the message “everything checks out” then you should name your new assembly and hit the green “Assemble” button.

Choose where to save your new molecule and Voila! You are done!

Question(s):

1. In the above example, which component is the vector and which is the insert?
2. When you combine a DNA insert and vector, which of these enzyme would we use? A) DNA polymerase  B) DNA ligase  C) Restriction enzyme
3. When preparing for cloning, how could you check that the restriction enzymes have produced the desired DNA fragments?

Practice:

Digest and Ligate Cloning - Manual
Can you recreate the pET-31b-JfyP construct by cloning manually and without the Benchling Assembly Wizard?

  • Identify where the ClaI and AlwNI restriction sites are in the pET-31b plasmid and ensure the JfyP gene insert possesses those restriction sites as well.
  • Copy the JfyP insert sequence and navigate back to your pET-31b sequence.
  • Create a copy of your pET-31b sequence and rename it to include the JfyP gene you want to clone in.
  • Now find the area between your restriction sites that your JfyP insert will be cloned into and paste it in.

Stretch yourself:

Follow along with these step-by-step guides and videos to find out how to carry out Gibson and Golden Gate through Benchling’s Assembly Wizard.

Gibson

Gibson assembly cloning is attributed to its creator Dr. Daniel Gibson who developed this method to join multiple DNA fragments through a single isothermal reaction. Each DNA fragment possesses overlapping sequence homology that is used to direct the assembly reaction. We’ll take a look at an example of Gibson cloning below where the JfyP gene is inserted into the pET 31b plasmid by designing sequence homology arms.

  • Open the “Assembly Wizard” and choose “Gibson” as your assembly method.
  • Set your backbone first using the pET-31b sequence. Identify and select a region of the vector that you will replace with your insert.
  • Now “right-click” to open a new menu and choose “Invert Selection” to highlight the portion of the vector you will keep and choose “Set from Selection” to finalize your backbone.
  • The Wizard assumes there is no existing homology between pET-31b and JfyP sequences and will create new homology arms for both that can be incorporated through PCR prior to Gibson Assembly. (Note: When using the Wizard, do not include pre-existing homology arms that you may have added manually)
  • Switch to the JfyP sequence and set the entire sequence as your insert. Name your assembly and hit the green “Assemble” button.
  • Verify the "assembly primers' that the Wizard created and click "Finalize" to generate your new plasmid and associated primers.

Golden Gate

Golden Gate cloning relies strictly on Type IIS restriction enzymes which are unique from traditional restriction enzymes as they cleave outside of their recognition sequence. Their cleavage leaves 4 bp flanking overhangs that are not incorporated into the recognition sequence which allow them to be customized for the direct assembly of various DNA fragments. The advantages of Golden Gate are similar to Gibson in that this method can be used for precise, scarless cloning. We’ll demonstrate how the same JfyP gene can inserted into the pET 31b plasmid using BsaI sites, a widely-used Type IIS restriction enzyme for Golden Gate.

  • Open the “Assembly Wizard” and choose “Golden Gate” as your assembly method.
  • Set your backbone first using the pET-31b sequence. Identify and select a region of the vector that you will replace with your insert.
  • Now “right-click” to open a new menu and choose “Invert Selection” to highlight the portion of the vector you will keep and choose “Set from Selection” to finalize your backbone.
  • Since pET-31b and JfyP sequences do not have BsaI sites for cloning, the Wizard will create directional BsaI sites for both that can be added by PCR prior to the Golden Gate assembly.
  • Switch to the JfyP sequence and set the entire sequence as your insert. Name your assembly and hit the green “Assemble” button.
  • Verify the "assembly primers' that the Wizard created and click "Finalize" to generate your new plasmid and associated primers.

Use the Benchling Notebook to plan and record cloning experiments


When setting up experimental cloning in the lab you will need to assemble your insert and vector DNA with ligase buffer, DNA ligase and water. You can use tables on Benchling to input formulas and automate volume calculations for the reagents in your ligation.

DNA fragments and plasmid maps all integrate seamlessly with your notebook. Try adding these to your notes as you map out a cloning strategy by dragging plasmid sequences directly into your entry or @-mentioning them.

Solution(s):

  1. JfyP gene is the insert and pET 31b is the vector
  2. DNA ligase
  3. You could use Benchling to generate virtual digests and compare the expected fragments produced against the ones you see after running a digest and gel electrophoresis in the lab.

Congrats! You've finished the learning module: Molecular Cloning Methods.

What’s next:

Continue on to our next molecular biology lesson on CRISPR Knockouts.

Or learn more about Benchling for Education.

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