This section of the tutorial describes how to correct things when Geneious Prime incorrectly selects the wrong restriction enzymes, or selects the wrong restriction digestion fragment for your ligation.
1. Select the pET26B vector and xynA PCR Product files again.
2. Go Cloning → Restriction Cloning to open the Restriction Cloning setup window.
3. Click on Enzyme Set and set the Enzyme set to the commonly used enzymes list.
The pET26B vector also has a single BglII site. BglII and BamHI have compatible cohesive ends. If you examine the regions and sites selected by Geneious for the cloning operation, you will see that it has incorrectly guessed the BglII site should be used for the cloning operation, resulting in a short (176 bp) BglII-NcoI fragment being selected for ligation.
4. To correct the site selection, right-click on the triangle on the pET26B tag and select Choose enzyme cut site for the 5' reaction.
5. Choose BamHI
from the list of available sites and go OK. This should restore
the correct sites and allow you to proceed with the restriction cloning
operation.
This concludes the Restriction Cloning Tutorial.