Restriction cloning of a multiple fragments in CLC tutorial

Go to parent Exercises for the Cloning tutorial

Locating the data to use

In a similar way you can insert multiple fragments in the vector. This time we want to clone AcGFP1 and Atp8a1 as a fusion in the pcDNA3 vector.

The GFP1 fragment was created in the exercise on simulating PCR and should already be present in the Cloning folder.

The Atp8a1 fragment still has to be generated: we cannot use the same fragment as in the exercise on cloning a single fragment because we need a different restriction site at the 5' end of the fragment. To be able to make the fusion with GFP1 we need a BspE1 site at the 5' end of the primer. The new forward primer was loaded in the Primers folder in the exercise on loading sequences into CLC. Try to do the amplification without peeking at the solution.

PCR amplify the Atp8a1 CDS using the new forward and the old reverse primer.

Expand Toolbox in the top menu, select Primers and Probes and then Find Binding Sites and Create Fragments Select the Atp8a1 mRNA sequence and click Next. Use the Browse button to select the two primer sequences and click OK. Leave the PCR parameters at their default values. Click Next and set the output options as previously. Click Finish. This generates a fragment table displaying the PCR product. Right-click the fragment and select Open Fragment. This will create a new sequence representing the PCR product. Save the sequence in the Cloning folder and close the views. You do not need to save the fragment table.

The pcDNA3 vector is stored as a .txt file in the /Documents/CLC folder. Try to import it without peeking at the solution.

Open the annotated sequence of pcDNA3 in CLC.

Click the Import button in the top toolbar Browse to the pcDNA3.txt file, select the file and click Next. Indicate the folder in the Navigation area where you want to store the new primer, in our case that's the Cloning folder in the Example Data. Click Finish. The vector now appears in the chosen folder in the Navigation area. You can double click it to open in the View area. Switch to the circular view of the vector by clicking the Show Circular View button in the bottom toolbar. Remove the 'ORF frame' annotations.

We will fuse AcGFP1 and Rab5 via the BspE1 restriction sites and clone the fusion in the pcDNA3 vector via the HindIII and BamHI restriction sites.

Simulating the cloning

Now you have all the sequences that you need for the cloning (vector, Atp8a1 and GFP1 fragment) and you know which restriction enzymes you are going to use.

Open the cloning editor
Expand Toolbox in the top menu, select Cloning and Restriction Sites and then Cloning. Select the Atp8a1 fragment, the GFP1 fragment and the pcDNA3 vector. Click Next. Ask to Open the result and click Finish.

This opens the cloning editor with the first selected sequence in view. First we have to create the fusion. When you take a close look at the fragment sequences you will see that the BspE1 restriction sites are not shown. You can create your own list containing the enzymes that are to be shown in the view.

Create an enzyme list containing the 3 enzymes we are going to use.
Expand Toolbox in the top menu, select Cloning and Restriction Sites and then select Create Enzyme List In the side panel expand Restriction Enzymes and click the Manage Enzymes button Select each of the the three enzymes by typing its name in the Filter box, selecting it and clicking the right-pointing arrow to add it to the list. Click OK and save the list in the Enzyme lists folder

Use the enzyme list in the cloning experiment.
In the side panel of the cloning experiment expand Restriction Enzymes and click the Manage Enzymes button. Select to use an existing enzyme list Click the browse button to navigate to the enzyme list that you created and use the right pointing arrow to select it. Use the right pointing arrow to select the 3 enzymes from your list to be shown on the view. Click Finish. Now you do see BspE1.

If you were to go to another view now, the changes you made in the side panel will not be saved. This means that you would have to set the choice of enzymes again the next time you view a sequence e.g. when you view the final construct. Fortunately you can save the settings in the side panel.

Save side panel settings.
To save the changes to the side panel, click the Save/Restore Settings button at the bottom of the side panel. Click Save Alignment View Settings This opens a window where you can type a name for these settings If you check Always apply these settings, these settings will be applied every time you open a view of a sequence.

Make a fusion of the two fragments.
Cut each fragments by selecting the two restriction enzymes while holding the Ctrl key. Once you have cut the two fragments the Stitch button becomes active. Click the Stitch button to create the fusion. This opens a window that shows the matching ends. In this window select Replace input sequences with result. Click Finish.

Indicate the part of the vector that you are going to use

At the top of the view switch between to the vector sequence (red). At this moment the vector sequence is annotated as a fragment not as a vector sequence. Annotate it as a vector sequence by clicking the Change to Current button (green) at the top of the Clone editor. Press and hold the Ctrl key while you click first the HindIII site and next the XhoI site. At the bottom of the view you can now see information about how the vector will be cut open. Since the vector has been split into two fragments, you can decide which one to use as the target vector. If you select one of the vector fragments, the corresponding part of the sequence will be highlighted.

Next step is to insert the fusion fragment.

Insert the fusion fragment.

At the top of the view switch to the fragment. Click the Add as Fragment button (red) at the bottom of the view to insert the fragment as it is (it was already cut in the previous step) When this is done, the Clone button (green) at the lower right corner of the view is active because there is now a valid selection of fragment and target vector. Click the Clone button to open a dialog to inspect the overhangs of the cut site, showing the vector sequence on each side and the fragment in the middle. Click Finish and the new construct will be opened.

You can save the resulting construct.