How do I simulate Gateway® cloning of multiple inserts in SnapGene?
SnapGene can simulate BP- or LR-type reactions, or a combination of BP and LR reactions, with 1 to 4 ordered inserts.
For your convenience, SnapGene provides a library of commonly used Gateway donor and destination plasmid vectors.
Gateway® is a registered trademark of ThermoFisher Scientific. See Gateway Recombination Cloning Technology to learn more about Gateway® Cloning.
Start the Gateway Cloning Tool
Click Actions → Gateway® Cloning → and choose the reaction, or combination or reactions, to simulate.
You can choose to perform BP cloning, LR cloning with one or more fragments, or BP + LR cloning with one or more fragments, in one operation.
In this lesson we will perform three-part Gateway Cloning and perform BP and LR reactions in one operation.
Define the First attB Fragment Source File
The "attB Fragments" tab for the first fragment is displayed when a cloning reaction is started.
Define the source of the first attB fragment by one of the following methods.
- Use the dropdown to open the fragment 1 source file.
- Drag and drop the fragment 1 source file into the window.
- Click the "Click here" link to browse and open the fragment 1 source file.
The Gateway tool does not automatically reorder the attB fragments so make sure you define the attB fragments in the order they will be assembled.
Define the Fragment Sequence
In Map view or Sequence view, click on a feature to select it as the source fragment.
Alternatively, in Sequence view, select a specific region of the sequence.
In the above example the "trpA1 UP' feature is selected as the attB fragment 1 sequence.
Define the Source and Sequence of the Remaining AttB Fragments
Switch to each "attB fragment:" panel in turn, define the source sequence file and the region of the source sequence, for each fragment.
In the above example the "GAL4' feature is selected as the attB fragment 2 sequence.
Define the First Donor Vector
Switch to the "Donor Vectors" tab and click to select the appropriate vector from the list of "Standard Donor Vectors". The selected vector will be displayed.
If you wish to use a "custom" donor vector then select the option "Custom Donor Vector X:" and use the dropdown menu to choose an appropriate donor vector from your sequence files.
SnapGene will automatically suggest the appropriate "multipart" donor vector specified by ThermoFisher for multipart Gateway cloning. Ensure you click on vector name in the list to select it, the vector will then be displayed in Map view.
In the above example, the vector "pDONR221 P1-P4" is suggested as the donor vector to create an Entry clone for part 1.
Define the Remaining Donor Vectors
Switch to each "Donor Vector:" panel in turn, and define the source sequence, and the region of the source sequence, for each fragment.
In the above example, the vector "pDONR221 P4r-P3r" is suggested as the donor vector to create an Entry clone for part 2.
Set the Destination Vector
Select the "Destination Vector" tab and choose the destination vector from the list of "Standard Destination Vectors".
If you wish to use a "custom" destination vector then select the option "Custom Destination Vector" and use the dropdown menu choose an appropriate destination vector from your sequence files.
In the above example, a custom plasmid pDESTHAW is selected as the destination vector.
Design attB PCR Primers
Select the "Expression Clone" tab and click Choose attB PCR Primers" to have SnapGene design appropriate PCR primers to amplify the insert.
- Set whether each insert is to be recombined in the forward or reverse orientation.
- Set the desired target melting temperature (Tm) for the new PCR primers.
Click Choose Primers to have SnapGene design appropriate primers for adding attB sites and amplifying each insert fragment.
View the Expected Expression Clone
Switch to the "Expression Clone" tab. The predicted Expression plasmid (created by PCR of inserts followed by BP reactions and an LR reaction), will be shown in Map view, the binding sites of the new attB primers will be displayed, the inserts will be colored alternately red and blue.
If you are generating in-frame fusions of translated features then switch to Sequence view to confirm reading frame fusions between inserts are as expected.
Name the New Primers
Switch to the "attB Fragments" tab to view the new primer sequences.
Switch to each "attB Fragment" in turn and name each primer (optional).
Name the Entry Clones
Switch to the "Entry Clones" tab.
Switch to each "Entry Clone:" in turn and enter an appropriate name for each Entry plasmid.
Name and Create the Expression Plasmid Sequence
Switch to the "Expression clone" tab and enter an appropriate name for the new "Expression" plasmid.
Click Clone to create all "Entry" and "Expression" plasmid sequences.
New unsaved Entry clone and Expression clone files will be created.
Click File → Save to save each new plasmid sequence to an appropriate location on your computer.
View the Expression Vector History
View the Expression clone sequence and switch to History view to see all new primers, all cloning steps and and all intermediate sequences generated during the Gateway cloning simulation.
Order Primers to Start Gateway Cloning
Export your attB primer sequences as text for ordering from an oligonucleotide synthesis service provider. See Export Primers for more information on how to export primers.