Opening the Gateway Cloning Modal
In order to perform in-silico gateway cloning, start by navigating to the “Cloning” tab in the top banner. Then select the “Gateway cloning” dropdown menu, here you will see the option to select BP or LR gateway cloning. Select the appropriate method and the cloning modal will open.
BP Gateway Cloning
To perform BP gateway cloning, select “BP cloning” from the “Gateway cloning” dropdown in the “Cloning” tab. This will open the BP cloning modal box. The first step is to select your donor vector, you will see an overview of all your library files on the left side of the modal under the tab “My sequence”, select the desired donor plasmid by clicking it and selecting “Open sequence” under the plasmid preview or simply by double clicking the file. Alternatively, you can paste in your desired sequence by navigating to the “Enter sequence” tab next to the “My sequences” tab on the left side. Here you can paste a sequence to create a new file and save the file to your library. Note that the donor vector must already contain aTTP1 and aTTP2 recombination sites.
Once you have selected the donor vector Photo51 will notify you if the file contains the required attP1 and attP2 sites. If you have selected the incorrect file you can change the donor file by navigating to the bottom right of the modal and selecting “Change vector” or by selecting the pencil icon at the top under the “Donor vector” tab. Once the correct selection is made select the “Next: Add attB insert” button at the bottom right to move onto insert selection.
You will now see the same file selection screen as before (notice at the top that you are now in the attB insert tab), use this to find the desired insert file or paste in the correct sequence. You can either select a file that already contains attB sites or you can select any file and subsequently simulate the addition of attB sites via PCR. If you have chosen a file that already contains attB sites then select “Use directly” otherwise select “PCR to add attB sites”. Select the region of your plasmid which you would like to insert or select the point at which you would like to linearize your plasmid via PCR. Once this is selected you can use the button “Create primers from selection” if you have selected a range or use the button “Create primers to linearize at cursor” if you have selected a linearization point. After selecting either option you will see that the generated primers contain the attB sites displayed in green. By default the primers are named “attB Insert.For” and “attB Insert.Rev”, these can be changed by clicking on the primer name. Once the correct sites have been added and the donor and insert are ready for assembly, a notification will appear at the top under the “Entry clone” tab that notifies you that you can proceed via the notification “Ready for Gateway cloning”. Once ready select “Assemble” at the bottom right of the modal box.
You will then see a preview of the final construct. If something is incorrect you can go back to the previous steps by selecting the tabs at the top. If you are satisfied with the final assembly select “Save assembled product” from the bottom right of the modal to save the new construct back into your library. Now the product is ready to be used as the entry clone for LR Gateway Cloning.
LR Gateway Cloning
To perform LR gateway cloning, select “LR cloning” from the “Gateway cloning” dropdown in the “Cloning” tab. This will open the LR cloning modal box. The first step is to select your donor vector, you will see an overview of all your library files on the left side of the modal under the tab “My sequence”, select the desired donor plasmid by clicking it and selecting “Open sequence” under the plasmid preview or simply by double clicking the file. Alternatively, you can paste in your desired sequence by navigating to the “Enter sequence” tab next to the “My sequences” tab on the left side. Here you can paste a sequence to create a new file and save the file to your library. Note that the donor vector must already contain aTTR1 and aTTR2 recombination sites.
Once you have selected the donor vector Photo51 will notify you if the file contains the required attR1 and attR2 sites. If you have selected the incorrect file you can change the donor file by navigating to the bottom right of the modal and selecting “Change vector” or by selecting the pencil icon at the top under the “Donor vector” tab. Once the correct selection is made select the “Next: Add entry clone” button at the bottom right.
You will now see the same file selection screen as before (notice at the top that you are now in the “Entry clone” tab), use this to find the desired insert file or paste in the correct sequence. Note for LR cloning the entry clone must contain attL1 and attL2 recombination sites, these cannot be added via PCR as they are too large compared to the attB sites for BP cloning. The process of BP cloning creates attL sites in the resulting clone, these clones can then be used for LR cloning, in this fashion gateway cloning is designed as a two step process. After you have selected your entry clone file by double clicking it or selecting it and pressing “Open sequence” under the plasmid preview, you will see a confirmation message on the left side notifying you that the file contains attL1 and attL2 sites. Additionally, a notification will appear at the top under the “Expression clone” tab that notifies you that you can proceed via the notification “Ready for Gateway cloning”. Once ready select “Assemble” at the bottom right of the modal box.
You will then see a preview of the final construct. If something is incorrect you can go back to the previous steps by selecting the tabs at the top. If you are satisfied with the final assembly select “Save assembled product” from the bottom right of the modal to save the new construct back into your library.
