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. 

precise_primers.png

 

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.

primer_manual.png

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.

 add_primer_configure.png

 

 

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Comments

  • Avatar
    Ariana Marcassa

    When I follow the tutorial no suitable primers are found for my ORF. I want to give Geneious a try but it is very frustrating to use this program, especially because I know I could just look at the sequence in Word and then have the primers synthesized! What am I supposed to do, continue to drop the optimal Tm until the program finds something it likes?

  • Avatar
    Hilary Miller

    If no primers are found by for a specific region its probably because the Tm is too high/low, or there are hairpins or high GC.  Relaxing the constraints on these parameters can help, or you can manually choose the region where you want to put your primer.  In Geneious R6 you can do this by selecting where you want to put your primer and going to Primers->Characteristics for Selection.  This will put a primer annotation on the region you have selected and calculate the Tm etc for that region.  

    In Geneious R7 you can manually create a primer by selecting the region, clicking "Add Annotation" and choosing "primer bind" as the annotation type.  You'll then be able to see the Tm and other primer characteristics before you create the primer annotation, and you can edit the binding site or add a 5` extension at the same time.