How do I design cloning primers?

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

To do this in R8 and Later, select the CDS you want to clone by clicking the annotation and go to "Design new primers".  In the drop down box next to "Task" select "Precise".  To design primers around the selected CDS annotation, tick the box next to "Included region".  

To do this in R6.1 and R7, select the CDS you want to clone by clicking the annotation and go to "Design new primers".  In the drop down box next to "Task" select "Cloning".  To design primers around the selected CDS annotation, tick the box next to "Included region".  

 To do this in R6.0 and earlier, select the CDS you want to clone by clicking the annotation. Next, choose “design new primers”, turn off the target region and turn on the included region (which should be the CDS). Change the product size to be the length of the CDS in both boxes since it must be exactly the same length as the CDS. Only generate 1 pair. Since you’re forcing the design to be in an area that may be less optimal you’ll also likely have to drop the Tm minimum setting. When you hit OK the primers should be designed exactly at the ends of the CDS.

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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.