User GuidesSnapGene User Guide Golden Gate AssemblyGolden Gate Cloning of a Single Fragment into a Vector

Golden Gate Cloning of a Single Fragment into a Vector

How do I simulate Golden Gate cloning of a single fragment into a vector?

Open or Import an Appropriate Vector Sequence

In this example we will import and use the NEB Golden Gate expression plasmid pGGASelect. - see this link for more information about this vector.

To import the vector, click File → SnapGene Online Sequences... and search for "pGGA".

Select the plasmid in the list and click Import. The sequence will download and open in SnapGene.

Start The Golden Gate tool

Click Actions → Golden Gate Assembly → Insert Fragment... to start the Golden Gate tool.

The tool will open showing the Vector tab. The open vector file will be loaded as the expected vector file for Golden Gate cloning. Use the dropdown (1) if you want to select a different vector.

By default BsaI will be selected as the Type IIS restriction enzyme for digestion. Use the dropdown (2) if you wish to use a different type IIS enzyme.

SnapGene will automatically select the correct restriction fragment for replacement by the insert (3).

If required specify the "Orientation of the vector:" using the buttons provided (4).

Define the Insert Fragment

Switch to the Fragment tab.

Specify the insert fragment by one of the following methods:

  1. Use the "Source of Fragment:" dropdown to browse and choose and view the sequence that contains the insert fragment.
  2. Drag and drop a sequence file into the window.
  3. Use the "Click Here" link to browse and open the insert sequence file.

To specify a feature as the insert, click on the feature in Map or Sequence View to select it.

Alternatively, switch to Sequence View to select the precise sequence region for insertion.

If appropriate Type IIS sites are not detected on the insert, SnapGene will automatically select the option to "Run PCR and then digest" with the enzyme.

Design Primers to Amplify the Insert and Add Appropriate Type IIS Enzyme Sites

Switch to the Product tab and click Choose PCR Primers....

Set the target Tm for the primers, set how many additional bases to include 5' of the Type IIS site then click Choose Primers.

SnapGene can add between 2  to 9 nucleotides 5' to the Type IIS recognition site. Addition of extra nucleotides may improve efficiency of digestion of the PCR product by the Golden Gate enzyme.

PCR primers will be designed with Type IIS sites that generate overhangs that are complementary to the overhangs generated by digestion of the vector.

The expected product generated by ligation of the digested vector with the digested insert will be shown in the Product tab.

Check the Fusion Boundaries

In the Product tab, switch to Sequence View and locate and check the vector-insert fusion points will create the expected product.

Rename the Primers (optional)

By default, SnapGene will name the newly designed PCR primers "Fragment.FOR" and "Fragment.REV".

Switch back to the Fragment tab to rename the newly designed primers.

Name and Create the Product(s)

In the "Create Product:" field, enter an appropriate name for the Golden Gate assembly product.

If you wish to also generate the intermediate Linearized Vector" and/or Fragment PCR products, then check each "Make File:" option and enter appropriate names for each file.

Click Assemble to generate the products.

All products will open as new unsaved sequences.

Click File → Save to save each file to an appropriate location on your computer.

View the Product History

Select the product file and select History View to see a summary of the Golden Gate cloning simulation.

Order the PCR Primers

Select the product file and select Primers View.

Select the two new primers and click Primers → Export Select Primer Data to export the primer information to a delimited text file.

Open the file in a text editor to allow copy and paste into an oligo synthesis service providers web order form.