Running Golden Gate assemblies with the combinatorial assembly tool

You can model Golden Gate assemblies and design constructs in bulk using the combinatorial assembly tool. With this tool, you can create visual representations of constructs and their sequence features, like annotations, and focus on relevant information by creating only the needed fragment combinations. Select existing primers for your assembly and carry over annotations from your constructs in the final assembly. Then, save a record of the assembly, including the fragments and primers used, as an object in Benchling.

This article explains how to access the assembly tool and how to use it to run a Golden Gate assembly.

Running an assembly requires several steps:

• Entering the tool
• Set up the assembly
• Configure bins
• Add fragments 
• Review and finalize constructs

Entering the tool

You can access the new tool either through the Assembly Wizard or by using the create options.

1. Assembly:
   • Look for "Combinatorial cloning."
   • If you're using legacy Assembly Wizard for Gibson or Golden Gate methods, you'll see a suggestion to try Benchling’s new tool.
2. Project and Global Create Menus:
   • Create > Assembly > Assemble DNA sequences by cloning.
   • Create > DNA / RNA sequences > Assemble DNA sequences by cloning.

1. Using the New Tool: Once you're in the new combinatorial tool, there's a banner at the top. This banner offers you the choice to stick with the new tool or switch back to the legacy bulk assembly tool if you prefer.

2. Switching Between Tools: If you decide to use the legacy bulk assembly tool, you'll also find a banner there. This banner makes it easy to switch back to the new combinatorial assembly tool whenever you want to

Set up the assembly

In the cloning tool, enter information about your assembly in the modal:

• Name: The assembly name
• Location: The project and folder the assembly will be saved to
• Number of fragment bins: The total number of backbone and inserts in the assembly
• Topology of construct: Circular or linear  
• Cloning method: Golden Gate, Gibson, or Homology. 

Additional parameters can be defined by:

• Type IIS Enzyme Selection: This is chosen at the assembly level, meaning the same enzyme is used across all fragments in the assembly.
• Fragment Production Method: Can be set globally for the entire assembly or can be adjusted individually for each fragment bin via Bin configuration.
• Primer Parameters: Established at the assembly level and are relevant only when primers are used.
  • Options for using primers ("Use a primer pair") must be selected in the fragment production method in the setup modal or for a specific bin to configure primer parameters.

Enter additional parameters specific to your assembly type at the bottom of the modal and click Save. 

Configure Bins

A new Assembly tab will open with Metadata, Overview and Constructs nested tabs. Then create bins or spacers to add your sequences to. First create bins or spacers and then add sequences. You have three options for preparing each bin:

1. Use Existing Cut Sites: Use existing Type IIS Enzyme cut sites.
2. Use a Primer Pair: Design or use existing primers to amplify your fragment via PCR, introducing overlaps for assembly.

To create additional Bins & Spacers: Click the + icon next to Bins & Spacers. Spacers can only be added next to bins where primers are used to amplify a fragment.

Add sequences 

1. Next to a bin, click the + icon and select your method for adding sequences. You can add open sequences, search for sequences using global search filters, or add sequences from a worklist. This opens a modal where you can interact with the sequence, linear, or plasmid maps.
2. In the modal, confirm the start and end site you wish to use for the assembly and the orientation. Select preferred primers, if needed.
3. Make adjustments to any other sequences in the bin, then click Add.
4. Repeat the steps above for additional sequences.

Tip: You can add and edit sequences directly in the Fragments table.  View a sequence in the table by clicking the row number and clicking Edit fragment.

Using existing primer oligos 

If you’re performing an assembly with primers to model PCR amplification, you can use primers you've already created as oligos. You can search for and select oligos in the Fragments table which has two optional columns for 3' and 5' preferred primers. You can specify one forward and reverse primer for a given fragment. 

If you add preferred primers, Benchling tries to use them to amplify the fragment and model the cloning reaction. If you don't add any preferred primers, primers are designed for you where relevant.

If the preferred primers you selected can’t be used – for example, the primer doesn't bind your fragment, introduce the appropriate cut site, or meet your melting temperature parameters – new primers are designed as part of the assembly and a warning displays. In some cases, your preferred primers might be used in one construct and new primers are designed for another. You can review the status of your primers during confirmation of your constructs.

Review and finalize your constructs

After selecting your fragments, you will:

1. Specify your constructs
2. Confirm your construct and resolve errors
3. Finalize and save your construct

Specify your constructs

You can specify your constructs two ways:

• Manually add rows and select your combination of fragments from the Constructs table. 
• Click Auto-populate in the Constructs table to generate all possible combinations of fragments. 

There is an option to skip bins for one or more fragments in a construct, by selecting the Skip option in the dropdown

Benchling will validate the junction for the fragments listed to the left and right of the skipped bin(s)

To delete constructs, right-click the row number in the Constructs table and select Delete construct. If any constructs produce a warning or are invalid, error messages display in the Status column. These need to be resolved before finalizing your construct.

Confirm your construct and resolve errors

After selecting your constructs in the table, click the Constructs tab to preview them. View the construct, its bases, and its annotations in more detail by clicking the diagonal arrows to expand the construct in a modal. If your assembly method involves primers, open the Primers tab to view the primers and their properties designed or used in the assembly. 

Finalize and save your constructs

To finalize your constructs:

1. Ensure all blocking errors are resolved, then click the Assemble button at the top right of the page.
2. Choose to create new sequences as needed:
   • Constructs (required)
   • Primers (optional, recommended if designed during assembly)
   • Fragments (optional, useful for saving fragments instead of PCR amplifying)
3. For each group of sequences (constructs, primers, fragments), select the following options:
   • Add to folder (required)
   • Add to worklist (optional)
   • Add to schema (optional; assigns a schema but does not register new sequences)
4. Access your finalized assembly from the project folder where it was saved.

Finalizing the assembly locks the assembly record, generates new DNA sequences for each construct, creates new primers where relevant, and populates the tables with references to these newly created sequence entities.