Create assemblies with the assembly wizard

Benchling has a variety of tools to support cloning and assembly, the assembly wizard tools are best used for single plasmids that use digest and ligate, Gibson, or Golden Gate assembly.The assembly wizard works by guiding you through a set of steps to designate your backbone, insert location, insert sequence, and generate a new set of primers for Gibson assemblies. 

If you do cloning design for many constructs at once, we recommend using the combinatorial cloning tool. 

Digest and ligate assembly 

1. Open a sequence, such as your insert or backbone, and click the Assembly dropdown in the lower right corner of the window and select Assembly wizard
2. Select Digest and ligate as your assembly strategy, then click Start
3. Navigate to your backbone sequence, in the sequence select all the bases except for the bases between your cut sites. To do this:
   • click the first cut site, hold the shift button, then click the next cut site
   • Right click and select Invert selection from the menu
4. In the Assembly wizard, click into the Backbone slot and then click Set from fragment
5. Now click into the Insert slot of the Assembly wizard
6. Navigate to your insert sequence, in the sequence select the bases between your two cut sites. To do this, click on the first cut site, then shift-click the second cut site
7. In the assembly wizard, click Set from selection
8. If you get an error message because the sequences didn’t match re-align the sequences by clicking Reverse orientation
9. Verify the preview by clicking the check
10. Once verified, you will provide a name for your assembly using the textbox at the right, then click Assemble
11. You will be prompted to provide a folder for the sequence to live in before it is created 

Golden Gate assembly

The steps for a Golden Gate assembly using the assembly wizard are the same as the digest and ligate steps listed above, except they use Type IIS enzymes. 

Gibson assembly 

1. Open a sequence, such as your insert or backbone, and click the Assembly dropdown in the lower right corner of the window and select Assembly wizard
2. Select Gibson as your assembly strategy, then click Start
3. Click into the Backbone slot and navigate to your plasmid sequence
4. In the sequence, linearize the backbone by clicking the cut site. Do this by holding the shift key and clicking again
5. Once the linearized backbone region is selected, click Set fragment
6. Click on the Insert slot and navigate to the insert sequence
7. Highlight all of the insert’s bases and then click Set fragment
8. Verify the assembled preview at left and provide a name for your assembly using the textbox at the right
9. Once the assembly is named and all errors are resolved, click Assemble
10. You will then have the option to define what folders the sequence and primers should go to before the assembly is created 

Frequently asked questions 

Q: Can I perform a Gibson assembly with fragments that are already digested? 

A: Our Gibson assembly wizard facilitates the fragment digestion as part of the assembly process. If you are looking to set up an assembly from digested fragments, we suggest that you use our copy and paste cloning method. 

Q: What is the ideal annealing temperature and primer melting temperature for Gibson assembly? 

A: The conventionally suggested annealing temperature is 50˚C. As a result, Tm minimum default is 50˚C. Generally, you will set your annealing temperature below or just below the primer Tm. 

Q: How does the Gibson assembly wizard factor in 5' and 3' overhangs? 

A: The Gibson Assembly Wizard performs assemblies based on the sense strand cuts (rather than overhang directionality) and designs primers accordingly, essentially combining the two sense ends. Depending on which kit/exonuclease is used, there would be different exonuclease activity (potentially 5' -> 3' or 3' -> 5'). We picked a common solution to cover both possible scenarios.

Q: Can I maintain restriction sites when using the Gibson assembly wizard? 

A: Our tool does not allow you to specify whether restriction sites are maintained in the assembly or not. We hope to build out this feature sometime in the future.