Primers Level Up: Part II

BY Sajith Wickramasekara
February 5, 2014
Today, we are making it even easier to design primers and manage your oligo libraries. You may remember we overhauled our primer system last October. However, we still weren’t completely satisfied and kept an open dialogue with dozens of users. These changes were over a month in the making and we’re excited to make them available to you.

Primer bases on sequence maps

Check your sequence map’s display options for a new addition: Primer Bases. Turning this on displays the actual oligonucleotide sequence inside the colored primer arrow.
Once you pick a 3′ binding location when designing a new primer, try typing in a few bases. You should immediately see the result on the sequence map. Any mismatches will be highlighted red and if an overhang is specified, it will clearly stick out.

Secondary structure analysis

We are now using the ViennaRNA package to provide secondary structure prediction for oligos. You can determine the minimum free energy monomers and dimers while editing an oligo, and all suboptimal structures can also be shown.

Easier oligo edits

You can now directly edit oligos while working with your sequences rather than having to duplicate first. If the oligo has be attached to any other sequence, the change will be visible there as well.
Primer pairs can also be simultaneously designed. Benchling shows you the melting temperature difference, product length, and minimum free energy heterodimer while you make edits.

Whether you are creating or editing, we try to keep you from creating duplicate data. As you modify your oligo sequence, Benchling will scan your entire library for matches. If it turns out you already have the oligo in your library, simply click “Use” to attach it without creating a duplicate entry. Likewise, if you are editing an oligo from your library that is attached to other constructs, Benchling will allow you to duplicate and modify it rather than potentially altering it in several locations.

Searching for oligos

Clicking Attach Existing in the primer design panel opens up a few powerful workflows:

  • Find all oligos from your library that bind to the current sequence. You can specify how many bases need to match, if mismatches are allowed, set temperature constraints, and even isolate your search to a specific region of the sequence.
  • Find oligo by name or exact bases makes it easy to find matches for a specific oligo you already have saved on Benchling.
  • Check for abitrary bases is useful for seeing if a string of nucleotides will bind anywhere on your current sequence.
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