How do simulate Golden Gate cloning of multiple fragments into a Golden Gate vector with appropriate Type IIS enzyme sites?
In this lesson Golden Gate cloning will be used to insert multiple fragments into a vector with preexisting BsaI sites.
Import or Open a Vector Sequence
Open or import the vector sequence with appropriate sites for Golden Gate cloning.
If required, set display of Golden Gate enzyme sites by clicking Enzymes → Use Enzyme Set → Golden Gate Enzymes.
Start the Golden Gate Tool
To start the Golden Gate tool, click Actions → Golden Gate Assembly → Insert Multiple Fragments....
Set the Type IIS Enzyme for Golden Gate Cloning
Click the "Golden Gate Enzyme:" dropdown menu to select the enzyme for the Golden Gate reaction. To select other Type IIS enzymes, see Set the Enzymes for Golden Gate Assembly.
SnapGene will automatically set the open file as the vector and display the vector tab.
If an appropriate pair of enzyme sites are present† they will be automatically selected and the option to "Digest with [the selected enzyme]" will be set.
†If the selected Enzyme does not have appropriate sites on the vector then see Golden Gate Cloning of Multiple Fragments into a Vector with no Golden Gate sites or Golden Gate Cloning into a Vector Cut with non-Type IIS Enzymes.
To select a different vector sequence click the "Vector:" dropdown menu and choose from the list of recent files, or click "Browse:" to locate a file on your computer.
Set the Number of Fragments for Insertion
Switch to the "Fragments" tab.
By default the Golden Gate tool starts expecting two insert fragments.
Click the +/- buttons to add or remove fragments. The number of fragments is displayed in the Tab Header.
For larger numbers of fragments, click the dropdown and choose "Number of Fragments". Enter the number of fragments and click OK.
Define the Insert Fragments Sequences
Note, if you are dragging multiple insert fragments into the fragment pane, you don't need to manually set the number of fragments. When you drop the files SnapGene will automatically create the required number of fragments.
To define the inserts use any combination of the following methods:
- Drag and drop files into the "Fragments" panel.
- Set the "Fragment" via the dropdown then click the "Source of Fragment" dropdown and choose a file from the list of Open or Recent files, or click Browse to choose a file.
- Click the "Click here" links to browse and choose one or more files.
Reorder the Fragments Sequences
If fragments are in the wrong order, click the dropdown and choose "Order of Fragments".
Select one or more sequences and use the arrows to change the order, then click OK.
Define the First Fragment Region
In the Fragments tab, if required, use the dropdown to select "Fragment 1".
In Map view select a feature defining the fragment 1 insert, or alternatively, switch to Sequence view and manually select a precise insert region.
In the above example we plan to generate an in-frame fusion between the xynB CDS and an mCherry CDS. Therefore, we switch to Sequence view and select the precise xynB sequence region, excluding the "TAA" stop codon.
The option to "Run PCR and digest with BsaI" will be automatically set as SnapGene assumes the need to amplify the selection by PCR. SnapGene will design primers to add appropriate BsaI sites to both ends of each insert fragment.
Define Other Fragment Regions
In the Fragments tab, use the dropdown to select each "Fragment" in turn.
In Map view, select the Feature defining the Fragment 2 insert region, or switch to Sequence view and manually select a precise insert region.
In this example, we click on the mCherry CDS feature to select it as the second fragment.
Design Primers to Amplify the Insert Fragments
Switch to the Product tab and click Choose PCR Primers....
Set the desired Target Tm for all PCR primers.
Set the number of nucleotides to insert upstream of the Type IIS enyzme site that will be added to each primer†.
Click Choose Primers to design PCR primers for amplification of all fragments that require addition of enzyme sites.
† SnapGene allows you to add 2 to 9 nucleotides 5' to the type IIS site. These additions are to ensure efficient digestion by the selected enzyme, see this useful NEB page for advice on enzyme cutting efficiency for sites close to fragment ends.
Confirm the Vector/Insert Fusion Points
In the Product tab, information about the predicted product is displayed.
- The predicted product will be shown in Map view.
- A list of all fragments will be shown in the right hand panel, showing the overhang for each fragment ligation.
- A schematic of the product is displayed, showing the overhangs for each fragment.
- SnapGene reports the predicted assembly fidelity in the bottom right panel. If SnapGene detects an issue with assembly, a warning or error will be reported.
Adjust Overhangs (Optional)
Click Adjust Overhangs... to change overhangs.
In this example, SnapGene is free to choose from a range of overhangs that can be used to for a scarless fusion between Fragment 1 and Fragment 2.
SnapGene will automatically choose the overhang with the highest predicted fidelity.
If required, choose an alternative overhang from the list of possible overhangs, then click OK to design new primers that will specify the selected overhang.
The Adjust Overhangs panel also allows confirmation of correct in-frame fusion of adjacent CDS reading frames.
View the Ligation Fidelity Details
Click the Details... link to view the Assembly Fidelity details.
A matrix is displayed showing predicted overhang interaction (mispairing) for all overhangs present in the Golden Gate Reaction.
Name the New Primers and Create the Product Sequence
Switch back to the Fragments tab, click the dropdown and switch to each insert Fragment in turn.
Edit the primer name fields and enter appropriate names for each newly designed primer.
Set an appropriate name for the Product sequence.
Click Assemble to create the Product sequence.
Click File → Save to save the Product sequence to an appropriate location on your computer.
When viewing the Product file, click the "Show colors" button in the side toolbar to color the inserts.
Switch to History view to see all steps simulated and all sequences/primers created during the Golden Gate Assembly procedure.
Order the PCR primers
See Export Primers for details on how to export and order the new primer sequences.