How do I design cloning primers?

When designing cloning primers it is often necessary for the primers to be exactly at the ends of the CDS. This is essential for when doing Gateway cloning for instance.

In Geneious R8 onwards, the Design New Primers tool has an option for designing primers precisely at the ends of a selected region.  To use this, select your CDS annotation, open Design New Primers and change the Task to Precise.  Tick the "Included Region" box and this should automatically be populated with the coordinates of your CDS.  Change the number of pairs to return to "1", and this will produce a single primer set that amplifies the entire CDS.  Any 5' extensions required can be added during the primer design by opening the Advanced tab in Design New Primers and specifying the required 5' extension, as shown in the screenshot below. 



In Geneious Prime it is also possible to manually create a new primer at a specific location on your sequence.  To use this feature, select the bases where you want the primer to bind.  You should see a floating Selection Hint that displays the length and Tm of the selected nucleotides. If you are satisfied with the position and Tm of the selection then you can click the Add Primer addPrimer16.png button to open the Add Annotation dialog.


The Add Annotation dialog will open with settings appropriate for creation of a new primer. You can then give the primer a Name, set the primer Direction and, if required, add a 5' extension.  Primer details, calculated by Primer3, are displayed in the Characteristics section of the dialog so that you can confirm the primer is appropriate for your intended application.




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