How to Simulate Restriction Cloning

Restriction enzyme cloning is a widely used method for inserting DNA fragments into plasmids using specific restriction sites and ligation. It involves digesting both the DNA insert and vector with compatible restriction enzymes, ligating the fragments together to form a recombinant plasmid, and introducing the construct into host cells for propagation.
On this page, you will find detailed instructions and examples showing how to plan and simulate restriction cloning with CodonCode Aligner.

The Restriction Enzyme Cloning Tool

Cloning with restriction enzymes is typically used when you have convenient restriction enzyme sites flanking your insert and vector, making it a simple, cost-effective, and reliable choice. The most difficult part of the cloning process is often planning it. The restriction enzyme cloning feature in CodonCode Aligner makes it easy to simulate and plan the process in silico. Virtual restriction cloning helps to find compatible enzyme cut sites, visualizes the ligation of vector and insert, and allows you to create the expected cloning product for clone verification.

This guide explains how to design restriction cloning in CodonCode Aligner.

Before simulating the cloning workflow, open an existing project in CodonCode Aligner, or create a new project, and add the sequences you want to use for cloning (for example by dragging the sequence files onto the project window).

To start the cloning wizard:

• You can choose the sequence you want to use as the vector now, or later in the cloning wizard. To choose it now, simply click on the sequence in the project view so it is selected.
• Choose Tools → Restriction Cloning... to start the cloning wizard.

This will open the cloning wizard with the sequence you selected as the vector:

Choosing Restriction Enzymes

The restriction cloning wizard starts with the Vector tab showing. If you have one sequence selected when initiating restriction cloning, this sequence will be pre-selected as the vector. If no sequence was selected, or you would like to use a different one, you can pick the sequence in the drop down box on the top right. Note that only sequences that are in your CodonCode Aligner project can be chosen in the drop down box.

The cloning wizard will use your current restriction enzyme settings to choose the cut sites that are displayed. The enzyme preferences can be changed directly in the cloning wizard using the enzyme button on the left side of the map:

Click on the button to select only enzymes that cut a specific amount of times, or to change the restriction enzymes to be considered.
For example, if your lab always has the same enzymes available, you can save a list with these enzymes, and use it to quickly have only these specific enzymes shown in your sequence map. Enzyme lists can be saved and selected by clicking on the Select Enzymes... menu option:

After selecting only unique cutters and just the enzymes from the "Popular Enzymes" list, the sequence map for my cloning experiment now shows fewer and better suited enzymes:

For a step by step example on how to change the displayed enzymes and save and load enzyme lists, look at the video tutorial Restriction Cloning.

Selecting Vector and Insert

In this example we would like to use the two enzymes HindIII and XhoI to cut both the vector and insert sequence. These enzymes were chosen because both sequences do contain unique cut sites for them which flank the regions we want to use.
The region can be selected by shift-clicking on the two enzymes, or by choosing the enzymes and fragment in the section to the right of the sequence map:

The overview at the bottom of the cloning wizard now shows a selection for the vector with the overhanging ends and enzyme cut sites.

To select the insert, switch to the Insert tab at the top of the cloning wizard. Then follow the same steps as when selecting the vector: Use the drop down box to choose the sample that contains the insert you want to use, and select the region that covers the insert area.
In this case I am choosing the insert called vaccine, and am again selecting the region between the enzymes XhoI and HindIII using shift-click:

The map layout can be switched between circular and linear maps using the button with the sequence map icon in the toolbar on the left.

The direction of the insert can be flipped by using the direction buttons below the insert in the overview at the bottom if your enzyme cut sites are reversed.

If the overhangs are not compatible, or either sequence has not been chosen, you will not see a valid product in the overview at the bottom, and the notice section at the lower right will not turn green.

The Cloning Product

Once you have chosen your vector and insert regions with matching restriction enzyme cut sites on either end, proceed to the Product tab:

Here you can double check your cloning product. The name for your cloning product can be changed at the bottom right. Clicking on the Clone button will create the product sequence in your CodonCode Aligner project.

You can either close the cloning wizard at this point, or you can leave it open, for example to repeat the cloning experiment with changes, like different restriction enzymes, or another insert sequence.

Cloning Result in your CodonCode Aligner Project

The resulting product sequence from the cloning workflow is added to your CodonCode Aligner project:

Viewing Restriction Cloning Warnings and Errors

The cloning wizard will help you spot and fix errors that can cause your cloning experiment to fail.

The most common cloning problems occur if incompatible enzymes are used. You can greatly reduce problems by using only enzymes that cut once - use the steps described in the section Choosing Restriciton Enzymes to change your settings.
If enzymes with incompatible cut sites are selected, the overview section in the cloning wizard shows a warning that the ends do not match:

If there are any problems, the text below the product will display the cause.

Non-matching cut sites are shown with a gap between them as opposed to matching cut sites which are displayed right next to each other. As a visual indicator, the yellow box on the right will also not turn green if you do not have a valid cloning product.

Note that cut sites can also become incompatible if the insert is reversed. Then the cut sites will not match even though the chosen enzymes are correct. In that case the insert can simply be flipped using the direction buttons shown below the insert in the overview.

Verification with Virtual Gels

If you are using agarose gels during any part of your cloning workflow, CodonCode Aligner also allows you to quickly create a virtual gel for comparison. In the example below we have made a gel for the cloning product created above, using the two enzymes from our cloning digest:

To simulate a gel, simply select the sequence in your CodonCode Aligner project and open a restriction view for it. Change the restriction view settings to display the virtual gel, and make sure the correct enzymes and comparison ladder are selected for the size of your sequence.

In the restriction view, mouse overs show the length and position of the gel fragments. You can click on any fragment, which will select the same region in your sequence in the base view.