Annotations and Tracks tab to the right of the sequence viewer and type COX1 into the filter box. In this exercise you will design PCR primers to amplify part of the COX1 gene from the mammoth genome.
Click on the Mammuthus primigenius (woolly mammoth) mitochondrial genome sequence and locate the COX1 gene. If you are having trouble finding COX1 display the Annotations and Tracks tab to the right of the sequence viewer and type COX1 into the filter box.
Once you have the COX1 annotation selected, Click the 
Primers button from the menu options and select Design New Primers.
In this dialog box you can specify where you want to put your primers, what size PCR product you want to return, and characteristics such as size and melting temperature. Under Task select Generic, and check the Included Region box. This specifies where the primers will sit, so should be set to 5,331 to 6,878 - the base pairs that the COX1 gene spans. If there is a specific part of the gene you want to amplify in the PCR, set this under Target Region. For this example we don't have a specific region of COX1 we want to amplify, but we want to ensure our PCR products are less than 300 bp long so that the primers will work on degraded DNA. So uncheck the Target Region box, and set product size to between 200 and 300 bp with an optimal size of 250 bp. Set Number of pairs to generate to 1.
The Tm Calculation section provides details on the formula used to calculate the melting point of oligos. You can leave this at the default settings.
Expand the Characteristics section. Here you can set the required properties of the primers, such as the length of the primer, the optimal melting temperature (Tm) and the penalties for hairpins and primer-dimers. For many applications the default settings will work fine, but you may need to adjust these if no suitable primers are found with the default settings, for instance if you have particularly GC or AT rich target sequence or the need for longer primers. For the primers we are designing here we can use the defaults for most of these options, with the exception of Max Tm Difference. Set this to 2 in order to restrict the Tm difference between F and R primers and ensure they will work in a single PCR reaction.
Your Design New Primers window should now look like this:
Click OK, and Geneious will now update the sequence with suitable primers displayed as annotations. Select the COX1 annotation and zoom in on it using the 
zoom button to the right of the sequence viewer. You should now be able to see primer annotations named with the base number they start at (if you cannot see them, make sure you have deleted COX1 from the filter box in the Annotations and Tracks tab). Click Save to save the primer annotations onto the sequence.
Place your mouse over the primer annotation and you will see a tool tip containing the characteristics of the PCR primer and product. You can see from this that these primers should amplify a product 250 bp long, and that the Tm for both primers is 60C.
Click on each primer annnotation, then click Extract to extract the primers to your document table. Give the primers a meaningful name - e.g. "Mammoth COX1_F" and "Mammoth COX1_R". They will then be available for use in other Geneious functions.
Exercise 2: Adding 5` extensions
Exercise 3: Testing saved primers
Exercise 4: Degenerate primer design
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