The Restriction Cloning window comprises 4 main sections.
The top section ➀ allows you to set your vector (Backbone), define how the Candidate Enzymes are chosen, and if chosen from an Enzyme Set, define which enzyme set to use.
The panel below this, referred to as the "Construct layout" panel ➁ shows tag representations of all sequences selected for the cloning operation. You can use the Choose... or Add Insert... buttons to add a new backbone or new insert sequences.
You can drag and drop tags to change their order, and click on the tag-associated triangle to see a dropdown menu that allows you to switch sites, reverse complement the sequence or modify digestion overhangs on each tag.
Any tag selected in panel ➁ will be displayed graphically in the "Detailed view" panel ➂. The region of the sequence that will be involved in the cloning operation will be highlighted by a thick blue line.
The bottom section ➃ provides options for how the Restriction cloning operations results are output.
You can click on the Help button
in the bottom left corner of the
window for more information on all of these options
The following steps describe how to perform restriction cloning using the example sequence documents provided with this tutorial.
1. Select the pET26B vector and xynA PCR Product files that are provided with this tutorial.
2. Go to Cloning → Restriction Cloning to open the Restriction Cloning setup window. pET26B should be selected as the backbone and shaded blue in the Construct Layout panel, and you should see "Use Leftmost" in the Backbone dropdown menu. You can also choose a specific document from this menu if it is not set correctly.
3. Set the Enzyme set to the pET26B MCS RE sites list. The Restriction Cloning tool should then correctly identify and select the NcoI and BamHI sites intended for this cloning operation. You can also see and select other restriction sites by clicking the down arrow on each document tab in the layout window
4. If you are satisfied the settings will correctly simulate your cloning operation, then go OK. A new file called pET26B - xynA_PCR_Product will be created.
Click the link below to go to Part 3: Confirming the CDS in-frame fusion.
Part 3: Confirming the CDS in-frame fusion
Part 4: Troubleshooting