Use primers

Benchling's tools for sequence primers allows users to design, verify, save, import, and export primers for DNA sequences. This feature streamlines the process of primer design and management, ensuring primers can be accurately mapped to sequences, linked in pairs, and used in other tools. The feature supports molecular biology workflows such as PCR, qPCR, and sequencing, as well as general DNA manipulation and cloning processes. There are two ways to design primers within Benchling: you can either do so manually or using the primer wizard.

Design primers manually

1. Open a DNA sequence and ensure you are in the Sequence Map view
2. Use the mouse to highlight and select your region of interest Right click on the region of interest,  and select Create Primer from the menu
3. Use the forward or reverse options to select the direction of your primer
4. This will open a new Design Primer tab that allows you to define other parameters for your primers

Design, verify, and save primers

The Design Primer tab in Benchling is a powerful tool for creating and analyzing oligonucleotide primers. You can introduce enzyme cut sites, automatically calculate and display important properties, and check for potential problems like hairpins or primer dimers. This tab allows you to visualize secondary structures and provides a Gibbs Free Energy (ΔG) value to help you assess stability. Once saved, primers  can be seamlessly used in other Benchling functions, such as the Assembly Wizard for cloning, and can be referenced in your electronic lab notebook for full traceability.

1. Decide if you want to create a single primer or pair. Use the single primer dropdown in the upper left corner of the tab to make the designation.
   • This will add the option to define both the forward and reverse strands of the primer, ensuring that you fill out both in order to save both primers 

2. To designate a reverse strand for primer pairs, either copy and paste bases in, or highlight the sequence in the Sequence Map and click Set from Selection in the Design Primer tab
3. To save your primer you will need to give it a name and assign it a folder however there is additional optional processing you can do on your primer:
   • If you want to add a restriction site to the primer, use the cut site dropdown to search for the enzyme restriction site you wish to add. The sequence will populate to the left, and you can copy and paste it into the Bases text box Add an overhand by using the arrows or typing in a number. As bases are added to the overhang they will be highlighted in grey

• To check for secondary structures, click Check Secondary Structure to investigate the structure(s) that form, use the temperature text box to adjust the temperature if needed. This will update the ΔG values in the section below. To view predicted structures, click on the Gibbs Free Energy (ΔG) values. Click View all structures to see a list of all possible secondary structures 

• To modify other thermodynamic parameters for priming, click on the Wrench icon

4. Once you’ve finished doing any additional processing give your primer a name and a folder to save to, then click Save Primer

Design primers with the primer wizard

Manually designing primers can be tedious and prone to error. It requires careful consideration of several factors, including melting temperature (Tm​), GC content, potential for self-dimerization, and off-target binding. The Primer Design Wizard in Benchling automates these calculations and checks, based on your input sequence and specified conditions. The wizard's step-by-step interface guides you through the design process, making it easy to input their desired sequence, set design constraints, and view potential primer candidates. 

By using the Benchling Primer Design Wizard, you can optimize and accelerate your primer design process.

1. Open your sequence and click the Primer icon on the Molecular Biology toolbar
2. Click Create Primers and choose Wizard from the menu

3. Use the Task dropdown to choose the primer type you want
4. Define the target region and adjust the other default parameters as necessary
   • To choose your target region you can select it on your sequence and click Use Selection
5. The sections of the Wizard help set your parameters
   • Region: Enter the start and end location for your primer or highlight it on the sequence map and click Use Selection
   • Primer: Specify min, max and optimal primer parameters for GC%, Tm , and Size. You may also set a 3’ GC clamp which is the strategic addition of one or two guanine (G) or cytosine (C) bases to the 3' end of a PCR primer to enhance the binding specificity and stability of the primer
   • Amplicon: Specify amplicon min/max size
     • The default amplicon length range is 200 - 1000bp. If your amplicon length is outside of this range, please adjust the defaults. If your amplicon length does not fit within the selected range, Benchling will indicate that "no primers could be found"
   • Result Generation: Specify the number of primer results you want returned
   • Partial Design: Specify part of the sequence that must be included
   • Design Across Junctions: Allows you to specify regions of minimum overlap to design primers across junctions
   • Force Location: Specify the exact nucleotide position where you want the 5’ and/or 3’ end of your primer to bind on the template DNA sequence.

6. When you are ready, click Generate Primers
7. This will open a new tab containing viable primers designed based on the given parameters and the Primer 3 algorithm
8. Choose your primer / primer pair of interest and click Save Selected Primers

9. If you want to download your selected primers for use outside of Benchling click Export as CSV

We use Primer3 to create primers in our Wizard. For more information on how parameters are used in generating primers, refer to the official Primer3 manual: https://primer3.org/manual.html

Bulk import primers

For scientists and lab managers who need to manage large collections of primers, the bulk import primers feature in Benchling is an essential tool. Instead of manually entering each primer one by one, this feature allows you to upload entire primer databases from a spreadsheet (like a CSV or Excel file). This can be useful when you are migrating from other software, standardizing primer collections, or managing high-throughput experiments. 

1. Click Global Create and select Import DNA/RNA oligos from the spreadsheet

2. Select the project folder where you want the primers to be stored, select the nucleotide type and click Next

3. From here you can paste all the primers as raw text or insert from a CSV spreadsheet. Then click Next
   • List sequence attributes in the following order, separated by commas or tabs: sequence name, bases in the sequence, and any description

4. Choose the correct column type based on column names from your spreadsheet or from your raw text

5. You can open your project and inspect your imported primers and if desired share your primers with other members of your lab

Access saved primers

Primers saved to projects can be used across multiple sequence entities). They can be mapped to sequences, linked in pairs, and used in other tools, like assembly and in silico PCR. 

1. Open Global search
2. Click the Type filter under the search bar and hover over the Entity option
3. From here select an Oligio schema from under the DNA or RNA Oligo section of the drop-down menu 

Attach and detach primers 

Attaching primers to sequences in Benchling is a powerful way to organize your molecular biology work, improve collaboration, and streamline your experimental design. It creates a direct, traceable link between the physical primer and the genetic template it's meant to bind. 

Benchling automatically tracks which primers are associated with which sequences, so you can easily see what a primer was designed for. It also allows you to perform in silico experiments such as a virtual PCR and visualization of binding sites. When you attach primers to a sequence and save them in a shared project or registry, your colleagues can easily find and reuse them.

To attach new primers to a sequence, follow the above section to create a primer manually or with the primer wizard. Primers automatically attach to the sequence they are created on. If you would like to view the sequence(s) a primer is bound to, you can access this in the Relevant Items tab of an oligo entity’s Metadata tab.

To attach existing primers to a sequence:

1. In a sequence, click the Primers icon in the Molecular Biology toolbar to open the Primers panel
2. Click Attach Existing to open the Find Binding Sites tab 

3. In the Find binding sites drop-down menu, select how you want to search for existing primers
4. Complete the search parameters:
   • You may add additional search locations (ex. multiple registries)
   • You must specify the nucleotide sequence type to search (DNA or RNA)
   • You may change the minimum number of matching bases and the degree of allowed mismatch in the sequences
5. When ready, click Find Binding Sites. Benchling will find all saved primers that meet the parameters you've specified
6. In the Primers Search Results that display at the bottom, select the primer(s) you want to attach
7. Click Attach Selected Primers in the top-right corner. A toast displays when the attachment is completed 

8. To detach primers from a sequence, open the Primers panel and select Detach. Alternatively, you can detach multiple primers by selecting Detach All Primers

Link and create primer pairs

You would want to link two primers on a sequence in Benchling to define a specific DNA amplification region, typically for a PCR (Polymerase Chain Reaction) experiment. This action creates a virtual PCR product, which is a powerful feature for validating your experimental design before you start in the lab. By linking a forward and a reverse primer on a template sequence, Benchling can simulate a PCR reaction and predict the exact DNA fragment that will be amplified. This allows you to confirm product size, validate the sequence, and check for unintended binding.

To link primers: 

1. Select the first primer, then press Shift and click to select the second primer
2. Right-click the highlighted region between the primers and select Link Primer

Alternatively, you can link primers from the Primers panel.

1. In the panel, click the Primer Pairs tab and click Link Primers

Unlink primers

1. In the Primers panel, click the Primer Pairs tab
2. Select the primer pair to unlink
3. Click Unlink

Note: Unlinked primers will remain on the sequence and can be re-linked if needed. 

Manage visibility of primers on sequences

You may want to view primers on your sequence map in Benchling to visualize their binding locations, orientations, and the specific region of DNA they are designed to amplify. This is a critical step for verifying your experimental design before you perform any wet lab work.

1. Click the gear icon in the sequence map toolbar
2. In the drop-down menu, check Primers
3. To display the primer bases on your sequence click the checkbox next to Primer Bases

Export primers from a sequence

1. Open your sequence and click the Primer button on the right side bar
2. Use the checkboxes to select all the primers you would like to export (use the checkbox at the top to select all primers)
3. Click Bulk Actions and use the Dropdown to select Download to CSV 

Export primers within a project or folder

You can export all of the oligos saved in a given project or folder.

1. Right-click on the folder containing the primers you'd like to export, and click Export.
2. Select the Oligos - Spreadsheet option in the Export Data modal. Click Export and your sequences will be downloaded to your computer.

Frequently asked questions

Q: How is primer melting temperature (Tm) calculated?

We have the option for two algorithms - SantaLucia or Modified Breslauer. You can adjust your preference by:

1. Navigating to the sequence containing the primer you want
2. Click on the primer. This should highlight the part of the sequence corresponding to the primer.
3. Clicking on Melting Temp on the bottom of your screen
4. In the Algorithm dropdown, select the algorithm that you would like to be used for melting temp calculations

Note: Changing the preference will change calculations globally across your account.