Try it! – Gibson cloning with a custom vector

The Vectors drop-down menu in the Project window is a shortcut to all of the vectors contained in the CloningVectors.sbp project, also known as the “Vector Catalog.” It is possible to add a custom vector to an open project, or to add it to the Vector Catalog for use with future projects. The first part of this tutorial deals with the former case, and takes you through the steps needed to add a custom vector to a particular Gibson cloning project.

You will then make the Gibson clone as verification that you added the custom vector correctly. Gibson assembly, offered by New England Biolabs, is a technique which allows up to 6 fragment inserts. When using Gibson assembly, restriction insert size must be relatively large in order to prevent primer overlaps that could keep the insert from being incorporated into the expression clone.

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.

2. Use File > Open to open the demo file Tn5w.sbd. The sequence opens in the Linear view.

3. Click on the neomycin/kanamycin resistance feature labeled aminoglycoside-3’-O-phosphotransferase to select that range of sequence.

4. Choose Cloning > Gibson, LIC Cloning to open the Project window.

5. Some of the cloning methods in SeqBuilder Pro have editable options. Click on the Preferences button to see the options available for Gibson cloning.

Leave all settings at the defaults and click Cancel to exit.

6. You’ll be using a custom vector that is not in the Vector Catalog, and therefore not in the Vector drop-down menu on the right of the Project window. To specify the custom vector:
   
   
   1. Go to the NCBI page for Promega cloning vector pSP73.
      
      
   2. Click on the Send to link near the top of the page, and make the following selections:
      
      
      
      
   3. Press Create File and then save the file when prompted.
      
      
   4. Use your file explorer to find the saved file, then drag and drop it on the Vector drop-down menu in SeqBuilder Pro’s Clone tab.

The vector is selected automatically, and a new Linearize button also appears.

7. Press Linearize to open the pSP73 vector in the Circular view. “Unique sites,” enzymes that cut the sequence only once, are displayed by default.

8. Use the Left Cut and Right Cut drop-down menus in the yellow bar at the bottom of the window to specify EcoRI and HindIII, respectively.

9. In the yellow bar, press Select.

10. Back in the Project window, press Try It and then Make It.

11. The Setup tab asks whether you wish to verify the clone. Press the Cancel button.

The Gibson 1 Clone 1 opens automatically in a new Document window. By default, the top pane shows the simulated clone in the Circular view.

This marks the end of the tutorial.