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.
Along the top left panel you can view a Mini Map of the Gibson Assembled plasmid (Figure 22.214.171.124).
- Below this, you can view the Assembly Summary which shows a breakdown of the options you selected throughout the wizard (Figure 126.96.36.199).
You can go back at any time and change or adjust any of your inputs.
- 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 188.8.131.52).
- A newly assembled plasmid will simultaneously be generated in the right panel and the newly inserted fragments will be highlighted (Figure 184.108.40.206).
You can also see the overlapping oligos as small triangles below the DNA sequence in circular and linear views.
You can zoom in to the sequence view to validate the overlap junctions and to visualize the primers on the sequence (Figure 220.127.116.11).
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 18.104.22.168).
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 22.214.171.124). 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.
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 126.96.36.199).
You can retrieve the Assembly Summary as CSV by choosing to "Export summary as CSV."
- 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.