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Tutorial: In silico cloning part I - finding the right enzymes
August 14, 2008
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Tutorial: In silico cloning part I - finding the right enzymes
Tutorial: In silico cloning part I - finding the right enzymes
In this tutorial, you will see how to insert a sequence fragment into a cloning vector and create a circular map of the vector. For this tutorial we wish to take the Atp8a1 gene which has previously been cloned into the pcDNA3 vector, and insert it into the pcDNA4_TO vector. This means that we first have to cut out the gene from the pcDNA3 vector and subsequently insert the fragment into the pcDNA4_TO vector as a very basic restriction cloning.
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The challenge of finding the right enzyme
The first challenge is to establish which restriction enzyme are suitable for cutting out the gene and inserting it again. A suitable restriction enzyme needs to fulfill three criteria: • It should have a cut site either right before or right after the Atp8a1 gene, so that we don't get too much of the vector sequence transferred to the new vector. • It should only cut the pcDNA3/Atp8a1 sequence once (then there is a smaller risk that we mix up the fragments). • The enzyme should be included in the list of enzymes that are readily available in the lab (you can construct your own customized list of enzymes for that - in this tutorial we use the list that is included in the example data as shown below). We would prefer to use the same enzymes for cutting out and inserting, so that the fragment would have ends compatible with the insertion site.
Opening the cloning editor
To get started, we need to get the two sequences into the Cloning editor ( ): select 'pcDNA3/Atp8a1' and 'pcDNA4_TO' from the Cloning folder | Toolbox | Cloning and Restriction Sites ( ) | Cloning ( ) | OK Now the two sequences are put into a sequence list which is shown in the Cloning editor as shown in figure 1. The small colored triangles represent restriction sites. Restriction sites for 10 different enzymes are shown per default, but we wish to consider a broader range of enzymes. First, go to the Side Panel, under Restriction Sites, and click the Deselect All button. This means that no restriction sites are shown in the view (you will notice the small triangles and the enzyme names have disappeared).
Find enzymes cutting inside selection
Now, we will let the Workbench find suitable enzymes for our cloning. First, make a selection right before the blue Atp8a1 gene annotation (between the green T7 promoter annotation and the gene, positions 883..975), and: right-click the selection | Show Enzymes Cutting Inside/Outside Selection ( ) P. 2
Tutorial: In silico cloning part I - finding the right enzymes
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Figure 1: Two sequences in the cloning editor: pcDNA3/Atp8a1 and pcDNA4_TO. This will show a dialog where tell the Workbench which enzymes should be considered. As mentioned in the beginning of the tutorial, we wish to use enzymes from an existing list of enzymes, hence: Click Use existing enzyme list | Browse for enzyme list ( enzymes' in the Cloning folder under Enzyme lists | OK ) | Select the 'Popular
In the panel below to the left you see all the enzymes from the list. Select them all and add them ( ) to the Enzymes to be used to the right (as shown in figure 2).
Figure 2: Selecting all enzymes from the Popular enzymes list. Click Next. In this step you can set the criteria for the enzymes to be shown. To the left, you can specify how many times the enzymes should cut inside the selection that you made (from 883..975), and to the right you can specify how many cut sites it should have outside the selection. The default settings is: 1 cut site inside the selection, and 0 cut sites outside the selection. And P. 3
Tutorial: In silico cloning part I - finding the right enzymes
this is exactly what we want: an enzyme which only cuts the sequence once right before the gene. Below there is a preview of the enzymes that fulfill these criteria. In our case, we have one enzyme: NotI (see figure 3.
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• ApaI • ClaI • NotI • XhoI
Figure 3: The NotI enzyme fulfills the criteria. When you click Finish, a NotI cut site should now be shown. What actually happens is that this enzyme is now added to the list of enzymes in the Side Panel (see figure 4).
Figure 4: The NotI enzyme is added to the Side Panel and the cut site is shown on the sequence. To find the enzyme to use for cutting at the end of the gene, repeat the procedure described in this section, but with this selection instead: 4629..4670 (right after the blue Atp8a1 gene annotation).
Investigating the results
Now you should have found four enzymes (one cutting before the gene and three cutting after the gene):
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Tutorial: In silico cloning part I - finding the right enzymes
When you look at the sequences you can see that three of these enzymes also cut the pcDNA4_TO vector sequence (see figure 5).
Figure 5: Zooming in on the cut sites on the pcDNA4_TO sequence.
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Since these cut sites are located right after the CMV promoter, they would be ideal for cutting the vector open to insert the Atp8a1 gene.
See how to cut out the gene and insert it in part two of the tutorial: "Tutorial: In silico cloning part II - inserting the fragment" which can be found at http://www.clcbio.com/tutorials.
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