This tutorial demonstrates how to cut and clone a signal transduction gene into the pENTR/D-TOPO vector.

  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 N.thermophilus_his.sbd. The sequence opens in the Circular view.
  1. Click on the PAS/PAC sensor signal transduction feature to select it.

  1. Choose Cloning > Clone Selected Fragment.
  1. In the Clone tab on the right, choose the Method named PCR-Directed Restriction Clone.
  1. In the Vector drop-down menu, click on the plus sign next to Gateway Vectors, then the plus sign next to Entry Clones and select pENTR/D-TOPO.

  1. Press the Digest button. A specialized version of the Circular view opens.
  1. In the yellow bar at the bottom of the view, choose the Left Cut site SacII and the Right Cut site AscI.

  1. Press the Select button. The Clone tab comes back into focus.
  1. Press Try it. Then press Make it.
  1. In the Project tab on the left, note the appearance of two new files: the Restriction insert and Restriction Clone.

  1. Double-click on the Restriction Clone file to open it in SeqBuilder Pro.

  1. Click on the History tab at the bottom of the SeqBuilder Pro window to see the Cloning Summary, including the steps needed to create this clone in your lab.

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