1.22.4 Step 2: Gibson Assembly: Select Backbone

Linearise Vector by Digest

  • Set the backbone by choosing the restriction enzymes that you would like to use to digest and linearise your vector (Figure 1.22.4.1).
  • By default, the "Popular" restriction sites preset will be displayed in the 5' and 3' restriction site lists.


    Figure 1.22.4.1: The ”Select Backbone” tab: digest.

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  • To configure your own preset to display, select "Configure" to open the restriction site dialog and select your desired sites (Figure 1.22.4.2). The 5' and 3' restriction site lists will be automatically populated with your preset. Single and double cutters in the destination vector will be shown in black and other enzymes which cut the vector multiple times will be grayed out.


    Figure 1.22.4.2: Restriction sites dialog for preset options.

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  • The enzyme preset will also be displayed on the vector sequence in the right panel.
  • Information regarding the buffers is displayed in a table (Figure 1.22.4.3).


    Figure 1.22.4.3: Table regarding buffer information for specific restriction enzymes.

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  • You can choose to "Regenerate" the 5' and 3' restriction sites in the destination vector, do nothing by choosing "Non Processed" and leave the sites unchanged, or "Eliminate" them (Figure 1.22.4.4).


    Figure 1.22.4.4: Choosing to regenerate the restriction site in the destination vector.

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