1.22.10 Step 4 - Gibson Assembly: Finalize

The "Finalize" step displays an Assembly Summary of your inputs and then allows you to "Run Assembly" to generate the assembled plasmid and the oligos.

Assembly Summary

  • Along the top left panel you can view a Mini Map of the Gibson Assembled plasmid (Figure 1.22.10.1).

    Figure 1.22.10.1: Mini map of Gibson Assembled plasmid.

  • Below this, you can view the Assembly Summary which shows a breakdown of the options you selected throughout the wizard (Figure 1.22.10.2).

  • You can go back at any time and change or adjust any of your inputs.

    Figure 1.22.10.2: Summary of Gibson Assembly of the plasmid.

Run Assembly

  • Select "Run Assembly" and a table of oligos and synthesized fragments will be generated. Here you can view the oligo sequences and other information such as which fragment the oligo overlaps, and which fragment it anneals to.

  • Oligos and synthesized fragments which violate the Assembly Parameters (see below for details) will be highlighted in red. Hover over the red sequence to view the warning and how to fix it (Figure 1.22.10.3).

    Figure 1.22.10.3: Error message for oligos violating Assembly Parameters.

  • A newly assembled plasmid will simultaneously be generated in the right panel and the newly inserted fragments will be highlighted (Figure 1.22.10.4).

  • You can also see the overlapping oligos as small triangles below the DNA sequence in circular and linear views.

    Figure 1.22.10.4: Assembled plasmid and Oligo Summary Table.

  • You can zoom in to the sequence view to validate the overlap junctions and to visualize the primers on the sequence (Figure 1.22.10.5).

    Figure 1.22.10.5: Sequence view showing overlapping oligos.

Changing Assembly Parameters

  • If you want to change the Assembly Parameters at any time, expand the "Assembly Parameters" section (underneath the Assembly Summary) and adjust the "Overlap length" and the "Target Tm for the overlap" (Figure 1.22.10.6).

  • For 2-3 fragment assemblies (including the backbone) a minimum overlap length of 15bp is recommended, although 20+bp overlaps will enhance the assembly efficiency. With 4-6+ fragment assemblies a minimum overlap length of 20bp is recommended. These defaults are assigned automatically according to number of fragments.

  • The Advanced Assembly Parameters allow you to choose which fragment you want to add the overlaps to. This could be useful when you do not want to modify all of the fragments in your assembly. By default, the overlaps will be split equally among the two fragments (Figure 1.22.10.6). Note: This option is only available between two PCR generated fragments.

  • Re-running the assembly again will generate the oligos according to these new parameters.

    Figure 1.22.10.6: Assembly Parameters and Advanced settings.

Save and Finish

  • You can choose to save your new assembled vector as a new project by selecting "Save final product." You will be prompted to choose a folder in the Material box to save the new project (Figure 1.22.10.7).

  • You can retrieve the Assembly Summary as CSV by choosing to "Export summary as CSV."

    Figure 1.22.10.7: Options for saving your final product.

  • You can also save the Gibson Assembly wizard, by clicking "Finish" so that you can conveniently re-enter the wizard again at any time to go back and adjust any step.