To Perform Click Cloning

Click Cloning can be used to duplicate or design cloning experiments in the lab. By performing a restriction enzyme analysis, selecting cut sites, digesting out a fragment and then ligating that fragment into compatible restriction sites in a vector sequence.

To perform a ligation

1. Open up the vector sequence that you want to clone into.
2. Open up the sequence that you want to clone into the vector.
3. Ensure Automatic Restriction Enzyme Analysis is turned on with the enzymes you want to digest with.
4. Check that there are suitable restriction sites surrounding the gene or fragment you need to clone. Unique enzyme recognition sites will be displayed in a different color to ones that will be cut twice or more. You may want to zoom into the MCS site to show only those sites. If the Enzyme file window is open then you can Select enzymes to see if these are present in both sequences.
5. Select a restriction site on one side of your fragment, hold down SHIFT and click on a site flanking your fragment
6. Select suitable flanking sites around the gene, by clicking on one, then holding down SHIFT and selecting the other. Please note that in circular sequences the selection will be made in a 5' to 3' direction.
7. Click DIGEST. The fragment will appear in the Cloning Clipboard.
8. Select the fragment in the Cloning Clipboard. Any compatible sites in the cloning vector will be highlighted.
9. Switch to the cloning vector window.
   • If there is an unambiguous ligation possible (i.e. a single compatible site for each of your fragment's ends highlighted in blue/green) then click Ligate
   • If there are multiple choices of sites for one or more ends select one site, hold down shift and select the flanking site.. Click ligate
   • If one or more sites are not compatible you will be able to modify the ends.

Alternatively you can drag and drop fragments to ligate them into a cloning vector:

1. Select two sites in your Cloning Vector
2. Drag the fragment from the Cloning Clipboard and drop it onto the Cloning Vector
3. The Ligation dialog will appear.
   • If the ends are compatible you can click LIGATE
   • If no ends are compatible then modify one or both ends until both ends are compatible then click LIGATE

You can also ligate a fragment into a single restriction site. This is a useful technique for removing a restriction site from a vector.

To remove a restriction site from a vector

1. Select the site that you want to remove from a vector
2. Click DIGEST
3. In the Cloning Clipboard click the fragment just digested and click CIRCULARIZE
4. Fill in both ends so that they are now blunt.
5. Optionally you may want to reset the origin of the new construct to reflect the previous one.

A new sequence window will appear which will match the original vector except this restriction enzyme will nbow be missing.

Instead of using the Automatic Restriction Enzyme Analysis you may want to use ANALYZE | RESTRICTION ENZYME ANALYSIS. This is more powerful and allows you to chose only enzymes that cut in a particular site (e.g. the MCS) or only enzymes that do NOT cut in the gene you want to clone.

Related Topics.

Click Cloning

End modification

Resetting the origin of a circular sequence.

Gateway and TOPO cloning

Restriction enzyme

Automatic Restriction Enzyme Analysis

Simulated Agarose Gel

Simulated Agarose Gel preferences