The tutorial Try it! – Create and edit primers for use in restriction cloning showed how to add restriction enzymes to a sequence. This tutorial demonstrates how to cut and clone the histidine kinase gene into the desired vector.
- Follow the steps in Try it! – Create and edit primers for use in restriction cloning.
- Select File > Open and choose the cloning vectors catalog, Lasergene ‘x’ Data/CloningVectors.sbp, where ‘x’ represents the version number.
- Resize and rearrange the two Project windows, as necessary, so you can see both at the same time.
- In the CloningVectors.sbp project, expand the Gateway Vectors folder, and then expand the Entry Clones folder.
- Find pENTR/D-TOPO and drag & drop it into the Vectors folder of your own cloning project.
- Open the PCR amplified insert from the Inserts folder by double-clicking on it. The sequence opens in the Linear view. Click on the Linear view to make it the active view, then click on the Enzymes tool ( ) to see that Unique Sites are displayed, by default.
- Click on SacII, to the left of the sequence, then Shift+click on AscI, to the right of the sequence. Notice in the header that the enzymes and the sequence between them are selected.
- Select Cloning > Copy Restriction Insert.
- Return to the Project window by selecting Window > Untitled Cloning Project 1.
- Double-click on pENTR™/D-TOPO from the Vectors folder. The sequence opens in the Circular view.
If you see a Synchronize Views message, click OK.
- Click on the Circular view to make it the active view, then select Edit > Go to Position. Type in 669..704 and click OK.
The region between the two recombination sites, shown in orange, is selected.
- Click on the Sequence tab to switch to the Sequence view. Notice that there are SacII and AscI sites spanning the TOPO recognition sites.
- Click on SacII and then Shift+click on AscI. The enzymes and the sequence between them are selected.
- Select Cloning > Copy Restriction Insert and then Cloning > Clone Selected Fragment.
- Click the Clone tab and use the Vector drop-down menu to select Vectors > pENTR/D-TOPO, the item from the Vectors folder in the Project tab on the left.
- Click the Try It button and then the Make It button. A new clone entitled Gibson 1 Clone 1 appears at the bottom of the Project tab. You now have a thermostable kinase within a Gateway entry clone, ready to clone into an expression vector.