General musings from the MacVector team about sequence analysis, molecular biology, the Mac in general and of course your favorite sequence analysis app for the Mac!

101 things you (maybe) didn’t know about MacVector: #43 – Compatible Restriction Enzyme sites have special highlighting

Have you ever wondered how you are going to clone a particular fragment into a specific vector? What destination restriction sites are compatible with the enzymes you’ve chosen? MacVector has a unique color-coding approach to make it easy to identify compatible sites. Here’s how it works;

First, select the source fragment you wish to clone. Simply click on the left hand enzyme in the Map view, then hold down the shift key and click on the right hand enzyme. The two sites and the intervening region will highlight. You can do this in the single sequence Map tab or in the Restriction Enzyme Results Map window;

APairOfHilitedRESites

Next, either click on the Digest toolbar button, or select the Edit | Digest menu item. This copies the fragment to the Cloning Clipboard;

Cloning Clipboard

Now, keeping the fragment highlighted in the Cloning Clipboard, switch to the Map view of a potential vector sequence. Again, it can be the Map tab of a single sequence window, or the Map results window from a restriction enzyme analysis;

PBR322BamHindHilited

Here you can see that the HindIII site has a pastel green background, indicating that it is compatible with the HindIII-generated sticky end at the right hand end of the selected fragment on the Cloning Clipboard. The BamHI site has a pastel red background, indicating that it is compatible with the sticky end generated by the BglII-generated sticky end at the left hand end of the fragment on the Cloning Clipboard. The highlighting is truly based on compatibility of the overhanging sticky (or blunt) ends – both BamHI and BglII generate 5′-GATC-3′ overhangs. If you now select the HindIII and BamHI sites, you can click on the Ligate button (or Edit | Ligate menu item) and the fragment will be inserted between those sites, creating a new construct.

MacVector is clever enough to notice that quite often there is only one possible way a selected fragment can be inserted into a target vector. In those cases, you don’t even need to select the sites – just choose Digest and the fragment from the Cloning Clipboard will be inserted into the vector. In those cases, you can even just drag the fragment from the Cloning Clipboard and drop it onto the Map tab and it will get inserted automatically. Go on, give it a try!

If the fragment on the Cloning Clipboard has identical sticky ends at both the left and the right end, then target sites in the vector get highlighted with a mix of half red and half green;

BamDoubleHilite

When you Ligate a fragment that can be inserted in either direction, the ligation dialog always gives you the option to “Flip” the fragment into the opposite orientation;

LigationFlip



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