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 18.104.22.168).
By default, the "Popular" restriction sites preset will be displayed in the 5' and 3' restriction site lists.
To configure your own preset to display, select "Configure" to open the restriction site dialog and select your desired sites (Figure 22.214.171.124). 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.
- 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 126.96.36.199).
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 188.8.131.52).
- Then select "Next" to start adding your fragments.