How to Simulate Blunt End Cloning

Blunt end cloning is a straightforward method for cloning DNA fragments that have blunt-ended termini, such as PCR products generated with high-fidelity DNA polymerases. This tutorial shows how to simulate a blunt-end cloning experiment in CodonCode Aligner.

The Blunt End Cloning Tool

The Blunt End Cloning tool in CodonCode Aligner lets you simulate the cloning of blunt-ended DNA fragments into compatible vectors. It guides you through selecting the insert and vector, performs the virtual cloning automatically, and generates the resulting construct for sequence verification and downstream analysis.

This guide explains how to design blunt-end PCR and TOPO 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 source for the fragment 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 → Blunt End PCR Cloning... to start the cloning wizard.

This will open the cloning wizard with the sequence you selected as the source for the fragment:

Selecting the Fragment

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

In this example we would like to use egfp as the fragment to insert into our blunt end cloning vector. The specific region for egfp inside the CVU55762 sequence can be selected by clicking on the feature itself, by using click and drag to select the desired bases / region, or by entering the base numbers in the section to the right of the sequence map:

The direction of the fragment can be flipped by using the direction buttons below the fragment in the overview at the bottom.

Selecting the Vector

To select the vector, switch to the Vector tab at the top of the cloning wizard. Then either choose one of the common commercial linearized vectors that come pre-loaded, or use your own custom linearized vector. If you do want to use a custom linearized vector, select the sequence from the drop down menu at the top right of the cloning wizard. Note that any vector sequence you wish to use must be linearized. The whole sequence will be used as the vector.

To linearize any circular sequence, open the Sample Information window , and de-select the Circular DNA check box. If you do want the linear sequence to start at a different position than the current base that is numbered base 1, you can set the base number before linearizing the sequence using the Set Base Number... feature from the Sample menu.

The overview at the bottom of the cloning wizard updates the selections for fragment and vector as you select the sequences. Here you can verify that the sequences have the correct length.

The layout can be switched between map and bases using the Map and Bases buttons below the sequence display.

The toolbar on the left side of the cloning wizard enables you to show or hide features, enzymes, the minimap at the top, and to change the map or base layout.

Designing Primers for Blunt End Cloning

To create primers select the Primers tab.
When you first switch to the Primers tab, and vector and fragment are defined, you will see a dialog prompting you to pick primers:

The dialog shows a summary of the regions to amplify and allows you to set a target melting temperature (Tm) for the primers.

Click on the Pick Primers button to automatically create the cloning primers that match your chosen settings:

Since you are using a linearized vector for blunt end cloning, the primers will only be designed for the fragment.

The primers, as well as the primer names, can be changed manually in the table that displays the primers. For example, if you do want to add spacers, click on the primer sequence to edit it.

Once all elements (vector, fragment, and primers) have been chosen and are correct, the cloning wizard shows a cloning product in the overview at the bottom and a green flag at the bottom right of the wizard.

The Blunt End Cloning Product

Once you have chosen the fragment, vector and primers, proceed to the Product tab:

Here you can double check your cloning product and choose which samples and files to create for downstream analysis. In the section on the top right of the cloning wizard, you can choose to create samples for the primers, linearized fragments, vector, and the alternate cloning product. If you choose to create these samples, they will be added to a folder in your CodonCode Aligner project along with the cloning product. You can also export any of these sequences as one or multiple files, which, for example, is useful for ordering primers.

Please note that blunt end cloning also creates an alternate cloning product with reversed insert orientation.

The name for your cloning product can be changed at the bottom right. Clicking on the Clone button will create a "Blunt_End_Cloning" folder with your cloning product and any sequences you chose to create 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 a different linearized vector, or another insert sequence.

Cloning Results in your CodonCode Aligner Project

The resulting product sequence from the cloning workflow, along with any other sequences you chose to create, are added to your CodonCode Aligner project: