1.22.8 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 PCR

  • If you select "Generate by: PCR" forward and reverse primers which are 20bp in length will be automatically added to the primer fields and will appear on your sequence in the right panel (Figure 1.22.8.1).


    Figure 1.22.8.1: Generate fragment by PCR.

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  • You can adjust the length of the primers and alter parameters such as the length, the Tm and the GC content.

  • 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 extract your fragment (Figure 1.22.8.2). The new primers generated from Primer3 will replace the previous primers in the primer fields and be displayed on the sequence.


    Figure 1.22.8.2: Primer3 auto design primer advanced settings dialog.

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