Opening the Golden Gate Cloning Modal
In order to perform in-silico Golden Gate cloning, start by navigating to the “Cloning” tab in the top banner. Then select the “Golden gate cloning” button and the cloning modal will open.
Selecting and Preparing the Vector
In the Golden Gate 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. For Golden Gate cloning you will see several options on the right side to prepare your vector file. First you can select the type of IIS restriction enzyme you would like to use. These are grouped into Golden Gate and Other type IIS enzymes, use the “Type IIS Enzyme” dropdown at the top left to make your selection. If you would like to use the vector directly you can set this by opening the “Linearize by” dropdown menu and selecting “Use directly”. Once you have selected your vector file and enzyme, you can select “+ New insert” from the bottom of the modal.
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 or use it directly if it is already linearized and prepared. You can digest you selected insert with the corresponding Type IIS Enzyme or you can select a region or point and automatically create primers with the correct overlaps via PCR. 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.
Completing Golden Gate 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.
