Opening the HiFi Assembly Modal
In order to perform in-silico HiFi assembly, start by navigating to the “Cloning” tab in the top banner. Then select the “Restriction cloning” button and the cloning modal will open.
Selecting and Linearizing the Vector
In the restriction cloning modal box you will first have the option to select your vector. By default the current open file will be selected, if you would like to select a different file as the vector navigate to the bottom right of the modal and select “Change vector” or use the pencil icon at the top under the “Vector” tab. This will allow you to select a new file from your library or enter a sequence via copy pasting. If your selected file is a circular plasmid, you must first linearize the sequence. Alternatively, for linear ligation of fragments you can skip the vector selection by using the “Continue without vector” button at the bottom right. On the left side you will see the option to select a “Linearize by” method, select the “Restriction digest” option or the “Use directly” if you already have a linear fragment with compatible ends. For linearization via restriction digest, first select the type of cutter (unique, dual, unique/dual or all) then use the “Select enzyme” dropdown to search and select the desired restriction enzyme. Then select any desired 5’ modifications (removing overhang, filling overhang or adding 5’ dephosphorylation). If you would like to digest with multiple enzymes, use the “+ Add enzyme button” to add another enzyme. Once you have finished selecting and linearizing your vector, select the “+ New insert” button at the bottom right to select your inserts.
Selecting and Linearizing the Insert(s)
After selecting “+ New insert” you will be taken to the insert selection, you can choose a file from your library under the “My sequences” tab and open it by double clicking or enter a sequence via copy and pasting in the “Enter sequence” tab. You can then linearize your insert or use it directly if it is already linear. Navigate to the “Linearize by” dropdown and choose between “Restriction digest”, “PCR” and “Use directly”. Linearizing via restriction digestions works identically how it is described above. PCR is also available to add restriction sites to your inserts. Start by selecting the region you would like to amplify and insert or the point where you would like to linearize. Then select “Create primers from selection” or “Create primers to linearize at cursor” respectively. Once the primers have been generated you can used the “Digest with” option for each primer to add the appropriate restriction site and simulate the digestion prior to assembly. After selecting the enzyme select the “Add site to primer” you will then see the restriction site added to the primer sequence. Once you have finished linearizing your insert you can repeat the process for the next insert by navigating to the top of the modal and pressing the + icon next to the current tab or by navigating to the bottom right of the modal and selecting “+ New insert”. Once you are finished adding inserts, you can complete the assembly by selecting the “Assemble” button at the bottom right of the modal. Note that the assembly button will remain gray and unclickable until the fitting primers have been created for all inserts.
Completing Restriction Cloning
After selecting “Assemble” you will see a preview of the final construct. If there are any mistakes you can always navigate back to the “Vector” or “Inserts” tab to make adjustments. Once you are finished use the “Save assembled product” button at the bottom right of the modal to save the final product to your library.
