Opening the TA Cloning Modal
In order to perform in-silico HiFi assembly, start by navigating to the “Cloning” tab in the top banner. Then select the “TA cloning” button and the cloning modal will open.
Selecting the Vector
In the TA cloning modal box you will first have the option to select your vector. Note, by default the current open file will be selected, if this is not a TA cloning vector or not the file you intended, you can 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 vector, Photo51 has curated a large set of commonly used vectors which you can select from, otherwise you can select a new file from your library or enter a sequence via copy pasting. Once you have finished selecting your vector, select the “+ New insert” button at the bottom right to select your inserts.
Selecting and Preparing 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 via PCR or use it directly if it is already linear with the correct ends for your TA/TOPO cloning. For linearization via PCR select the type of polymerase, typically for TA/TOPO cloning you will need one that generates 3’ A/G overhangs, this is the default selection. Further, select a point to linearize or a region you would like to amplify / insert, then select the “Create primers to linearize at cursor” or “Create primers from selection” respectively. This will automatically create the primers based on the target annealing temperature set. You can add 5’ modifications or restriction sites to your primers if desired. Once you have finished linearizing your insert via PCR, you can complete the assembly by selecting the “Assemble” button at the bottom right of the modal.
Completing TA 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.
