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- Open all of the files containing the component parts.
- Change “display setup”, if necessary, to ensure that all restriction enzymes to be used in the cloning are displayed.
- Starting with the insert, open the file containing the insert sequence. In the graphic pane, click on the restriction enzyme site that is located at the left end of the fragment to be cloned. Holding the Shift key, click on the restriction enzyme site that is located at the right end of the fragment to be cloned.
- Using the menu at the top of the screen, click on Add fragment to Goal List. (If this was done correctly, you should just have to click on Next until the Add to List is displayed).
- Open the file containing the vector sequence; in the graphic pane, click on the restriction site that is compatible to the last enzyme chosen in step #3. Holding the Shift key, click on the other restriction enzyme site. Make sure that the correct piece of the vector is chosen in the display.
- Click on Add fragment to Goal List.
- If additional fragments are to be included in this construct, add them using the same procedure.
- Using the icon at the top, Open Goal list. The fragments that you added should be in the list. If more than two fragments are included in the construction, make sure that they appear in the order in which you want them to ligate to each other. The order can be changed at this time.
- Click on Run. You will be prompted for a name for this molecule and, if the fragments were entered correctly, the construct should be successfully created and will be shown in a new file.
- If you wish to change the starting coordinates of the newly created file, go to Molecule, Operations, Advanced DNA/RNA, Change starting coordinates.
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