This tutorial demonstrates how to cut and clone a signal transduction gene into the pENTR/D-TOPO vector.
- 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.
- Use File > Open to open N.thermophilus_his.sbd. The sequence opens in the Circular view.
- Click on the PAS/PAC sensor signal transduction feature to select it.
- Choose Cloning > Clone Selected Fragment.
- In the Clone tab on the right, choose the Method named PCR-Directed Restriction Clone.
- 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.
- Press the Digest button. A specialized version of the Circular view opens.
- In the yellow bar at the bottom of the view, choose the Left Cut site SacII and the Right Cut site AscI.
- Press the Select button. The Clone tab comes back into focus.
- Press Try it. Then press Make it.
- In the Project tab on the left, note the appearance of two new files: the Restriction insert and Restriction Clone.
- Double-click on the Restriction Clone file to open it in SeqBuilder Pro.
- 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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