1.22.9 Step 3: Gibson Assembly: Select Fragments

You then need to choose how to generate the fragments, either "By PCR" or "By Synthesis."

Generate Fragments by Synthesis

  • If you select "Generate by: Synthesis" then you will order your fragment synthesized from a DNA synthesis provider.
  • You can choose to codon optimize the insert by selecting the host organism from the drop down menu (Figure 1.22.9.1).


    Figure 1.22.9.1: Codon optimize the insert by selecting a host organism.

    </div>


  • The default setting assumes you will be synthesizing your fragment with the overlapping regions (Figure 1.22.9.2). In the final assembly an additional oligo will not be generated to assemble this fragment with an adjacent fragment.


    Figure 1.22.9.2: Default setting to synthesize overlaps.

    </div>


  • If you want to re-use this fragment for another assembly for example, then most likely you will not order the overlaps synthesized with the fragment. Therefore you should select the "No" option for "Synthesize Overlaps" and design primers to add the overlaps separately. The same interface of primer options will appear again where you can choose to optimize primer design with Primer3 (Figure 1.22.9.3).


    Figure 1.22.9.3: Designing primers after choosing "No" on synthesizing overlaps.

    </div>