1.22.5 Step 2: Gibson Assembly: Select Backbone

Linearise Vector by PCR

  • Set the backbone by typing coordinates into the "Start" and "End" fields. Or select sequence in the right panel and then either "Use Selection" or "Invert Selection" (Figure 1.22.5.1). The coordinates of your selection will automatically be added into the fields. You can switch views in the right panel to zoom into the sequence and adjust the caret to fine tune your selection.


    Figure 1.22.5.1: Linearise by PCR.

    </div>


  • Once you have set your selection, forward and reverse primers which are 20bp in length will be automatically added to the primer fields (Figure 1.22.5.2) and will appear on your sequence in the right panel. You can adjust the length of primers and alter parameters such as the length and the Tm.


    Figure 1.22.5.2: Primers for selection in Gibson Assembly.

    </div>


  • Alternatively you can optimize the design of your primers by selecting "Auto design (with Primer3)." You can adjust some general and advanced parameters to ensure you design the exact primers you need to linearise your fragment. The new primers generated from primer3 will replace the previous primers in the primer fields and be displayed on the sequence.

  • You can view the primer properties in the "Verification" display, as well as viewing primer secondary structure by clicking "IDT Oligo Analyzer" (Figure 1.22.5.3).


    Figure 1.22.5.3: Primer properties in the Verification tab.

    </div>


  • Then select "Next" to start adding your fragments.