MultiSite Gateway Pro technology enhances the classic Gateway cloning protocol by allowing you to clone up to four regions at one time. In MultiSite Gateway Pro cloning, three additional types of recombination sites have been engineered to recombine uniquely between the two ends of a source sequence and a host vector.

In this tutorial, you will create a clone that has a Pol promoter, two multiple cloning sites, and a reporter gene.

  1. If you have not yet download and extracted the tutorial data, click here to download it. Then decompress (unzip) the file archive using the method of your choice.
  1. Use File > Open to open the file MultiSiteExample.sbp in the Project window.
  1. Click on the plus sign next to Inserts to expand the folder.

  1. Choose Cloning > Multisite Gateway Pro Cloning.
  1. Use Shift+click to select all four inserts, then drag and drop them in the Fragments area to the bottom right of the Clone tab
  1. Reorder the fragments to match the image below. To do this, click on a fragment to select it, then drag and drop it in the new position.

  1. In the Clone tab, click on the Vector drop-down menu and select pDEST8.
  1. Click Try It and then Make It.
  1. The Setup tab asks whether you wish to verify the clone. Press the Cancel button.

In the Project tab, a new folder is added containing four amplified inserts (all linear), as well as four entry clones and the final expression clone (all circular).

The Multisite Gateway Pro 1 4-insert Expression Clone 1_ opens automatically in a new Document window. By default, the top pane shows the simulated expression clone in the Circular view.

  1. Click on the History tab at the bottom of the window to open the History view, which contains the Cloning Summary. The summary includes a nine-step cloning procedure and lists the primers needed for each step.

To print the Cloning Summary or copy it to the clipboard, see View and print cloning results.

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