Restriction cloning is a method of editing genes by cutting them with restriction enzymes at restriction sites. The cut genes are then joined with a method known as ligation. This post will show an example of how to use SnapGene for restriction cloning using one of the protocols available in SnapGene <insert link to first post>.
The first step in this process is to select an enzyme set based on your protocol. To do that, click on choose enzyme set, which is the drop-down arrow next to the show enzymes button on the left, and click on unique cutters or unique and dual cutters. Unique cutters will highlight enzymes that cut the sequence in only one place, while unique and dual cutters will show unique enzymes that cut the plasmid in two places. Hovering your mouse over any of the enzymes will show its dual.
Let’s now create an example by inserting GFP into a plasmid with chloramphenicol resistance. We will start by importing the plasmid by clicking on File -> import -> SnapGene online sequence -> Select pSB1C3 in the popup and click on Import. We should now be able to see the plasmid in the window as in the image below. The details of the plasmid can also be seen in the pane on the left side of the window.
The next step is to insert some bases into the sequence of the GFP to introduce new restriction sites that do not cut within the GFP itself. To do this, copy the biobrick prefix from the plasmid using the Sequence view by clicking View and selecting Sequence.
Next, open the file containing the GFP sequence and click on the beginning of the sequence. Then click on Edit, go to Insert, and select Bases. Paste the sequence for the biobrick prefix in the textbox and click on Insert. Now repeat the same process for the suffix and insert it at the end of the GFP sequence. At the top right, select EcoRI and PstI as your restriction enzymes.
The vector (target) of our cloning, in this case, is our plasmid. We will, therefore, perform the following actions on it to insert the GFP sequence. Start by clicking on Action, go to Restriction cloning, and click on Insert fragment.
Now click on Insert at the top of the window to go to the Insert tab and select Insert from the source from the drop-down at the top-right. Select the GFP file if it is available, or select the Browse option at the bottom of the list and navigate to its location. Once again, select EcoRI and PstI as your enzymes, and click on Clone at the bottom-left to perform your cloning.
That’s it! You’ve successfully inserted the GFP gene into the pSB1C3 plasmid. There are a number of alternatives to this method (such as adding restriction sites directly instead of inserting the biobrick prefix and suffix), and each method comes with its pros and cons. A bit of exploration will help you find the method that suits your needs.