Restriction cloning of a single fragment in CLC tutorial

Go to parent Exercises for the Cloning tutorial

Locating the data to use

Open the Example data folder in the Navigation Area. Double-click the ATP8a1 mRNA sequence and zoom to Fit Width (red) and you will see the light green annotation which is the CDS of the gene (green). This is the part that we want to insert into the pcDNA4_TO vector.

Expand the Cloning folder in the Navigation Area. Double-click the pcDNA4_TO sequence (red). In order to see which restriction enzymes can be used, we create a split view of the vector and the insert:

• Right click the pcDNA4_TO tab (green)
• Select View
• Select Split Horizontally (blue)

For the vector sequence switch to the Circular view (red) and zoom in (green) on the multiple cloning site downstream of the green CMV promoter.

Restriction analysis of the vector

Find two enzymes from the multiple cloning site in the vector that do not cut in the Atp8a1 gene.
By looking at the enzymes we can see that for instance HindIII and XhoI cut in the multiple cloning site of the vector and not in the Atp8a1 gene.

Which overhangs are created by these enzymes ?
Hovering your mouse over the name of an enzyme shows extra info but it does not show the ends it creates as in SnapGene: You can see the ends that are created by these enzymes by expanding the 'Restriction Sites (red) in the Side Panel. Click the Sort enzymes by overhang type button (green) and place your cursor over HindIII in the Side Panel. You will see how the enzyme cuts.

Since the restriction sites are not found in the atp8a1 gene, we have to add them to the primers we are going to use to amplify the Atp8a1 CDS.

Add a HindIII restriction site to the forward primer

Close the views of the vector and the mRNA sequence and open the ATP8a1 fwd primer sequence from the Primers folder. Double-click the name of the sequence to make a selection of the full sequence. If you do not see the whole sequence turn purple, please make sure you have the Selection Tool chosen. If the sequence is selected, right-click and choose Insert Restriction Site Before Selection (red) In the Filter box enter HindIII (red) and click it (green). At the bottom of the dialog, add a few extra bases 5' of the cut site (blue ; this is done to increase the efficiency of the enzyme). Click OK and the sequence will be inserted at the 5' end of the primer

Exercise: Add a XhoI restriction site to the reverse primer

Perform the same process for the ATP8a1 rev primer, this time using XhoI instead. You should also add a few bases at the 5' end to efficiency of the enzyme.

The ATP8a1 rev primer is designed to match the negative strand, so the restriction site should be added at the 5' end of this sequence as well (using Insert Restriction Site before Selection). The end result should be the following:

Save the two primers and close the views.

More on restriction analysis in CLC

As far as we know, CLC does not offer you the option to find enzymes that generate compatible ends as SnapGene does.

Check which enzymes cut only once in the atp8a1 mRNA
CLC shows by default all cutters that cut regardless of how many times they cut in the sequence (red). If you want to change this expand 'Restriction Sites in the Side Panel. Deselect all cutters except the Single cutters (green):

Check which enzymes cut only once in the atp8a1 mRNA and create a 3' overhang

Expand Toolbox in the top menu and select Cloning and Restriction Sites and then Restriction Site Analysis (red): Select the ATP8a1 mRNA sequence and click Next Select Use existing enzyme list and select Popular enzymes (red). Write 3’ into the left filter (green), select all enzymes generating 3' overhangs(blue) and click the Add button (purple): Click Next. In the next step you specify that you want to use enzymes that cut the sequence only once: Select the One restriction site checkbox and click Next Select Add restriction sites as annotations on sequence and Create restriction map. Click Finish You see that there are only two enzymes that fulfill these criteria: SacII and KpnI.

Simulating PCR

Before we can start the cloning we first have to generate the fragment that we are going to insert by simulating PCR.

PCR amplify the Atp8a1 CDS using the forward and reverse primer designed previously.

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. The Match criteria determine how well a primer should match the sequence. Exact match specifies that all positions of the primer must base pair with the sequence. Note that we cannot choose this option because we have added non-matching restriction sites to the primers. You can also adjust Concentrations of the reaction mixture. This is used for calculating melting temperatures of the primers. Click Next and adjust the output options 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.

Exercise: Simulate PCR of pAcGFP1-C1 with the GFP primers

In the /Documents/CLC folder you find two .clc files containing the primer sequences to amplify GFP1 from the pAcGFP1-C1 vector. Import the primer sequences into the Primers folder.

The PCR product of GFP1 will be fused to Atp8a1 via the BspE1 restriction sites and the fusion will be cloned in the pcDNA3 vector via the HindIII restriction site. This is why the forward primer contains a HindIII restriction site. At the 3' end the GFP1 gene already has a BspEI restriction site for in frame cloning of target genes, so the reverse primer is just a regular primer chosen right downstream of the BspE1 site.

Try to solve the exercise without peeking at the solution.

PCR amplify GFP1 using the forward and reverse GFP primers.

Expand Toolbox in the top menu, select Primers and Probes and then Find Binding Sites and Create Fragments Select the pAcGFP1-C1 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 adjust the output options 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.

Simulating the cloning

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

Open the cloning editor

Open the cloning editor
Expand Toolbox in the top menu, select Cloning and Restriction Sites and then Cloning. Select the Atp8a1 fragment and select the pcDNA4_TO cloning vector in the Cloning folder. Click Next. Ask to Open the result and click Finish.

This opens the cloning editor with the Atp8a1 fragment in a linear view.

Prepare the vector sequence

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

At the top of the view you can switch between the sequences used for cloning. Switch to the vector sequence. 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 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. The information is not as clear as in SnapGene. 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.

In CLC you select the part of the vector you are going to keep while in SnapGene you select the part of the vector you are going to replace !

Prepare the insert

Next step is to cut the fragment.

Indicate the fragment that is going to be inserted.

At the top of the view switch to the fragment. Perform the same selection of restriction sites as before while pressing the Ctrl key. At the bottom of the view you can again see information about how the fragment will be cut. Since the fragment can split into two fragments, you can decide which one to use as the target fragment. If you select one of the fragments, the corresponding part of the sequence will be highlighted.

Perform the cloning

Perform the cloning.
When this is done, the Clone button 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.

Save the resulting construct.

Showing the cloning process

Go to the pcDNA4_TO_Fragment... sequence: this is the product generated by cloning the Atp8a1 gene in the pCDNA4_TO vector.

Clicking the Show History button in the bottom toolbar shows how this sequence was generated:

The history is not as nice as in SnapGene.