Access free primer design tools for PCR, cloning, and Sanger sequencing

OligoPerfect Primer Designer

Screenshot of OligoPerfect primer designer tool dashboard

Invitrogen OligoPerfect Designer is a convenient primer design tool for PCR, CE sequencing, and cloning. This free and user-friendly cloud-based software is built on the trusted Primer3 algorithm, ensuring accurate and efficient primer design for your experiments.

With OligoPerfect Designer, you can easily upload up to 50 DNA template sequences, allowing you to tailor your primers to your specific reaction conditions. This can help save you valuable time and effort, ensuring optimal results every time. Plus, OligoPerfect Designer allows for easy download of sequences that can then be ordered through our convenient online ordering system.

Using OligoPerfect Designer is a straightforward process. Simply paste or load your FASTA-formatted sequences, select your desired parameters, and let the software do the rest. Choose from a list of available primers and easily add modifications as needed. You can also select the purification method, format, and other options before adding the primers to your cart.

Not ready to order just yet? No problem. OligoPerfect Designer allows you to save your project, so you can easily return to it whenever you're ready.

Design your primers

NOTE: OligoPerfect requires you to sign in to Thermo Fisher Cloud. You will be directed from the link above to the sign in page, where you can create an account if needed.

Primer Designer Tool

Creative imagery of a primer on a DNA strand

The Primer Designer Tool lets you quickly search for, configure, and order primers for the Sanger confirmation step of your NGS workflow. The database consists of ~650,000 pre-designed primer pairs for re-sequencing the human exome and human mitochondrial genome.

  • Our primers are free of SNPs and primer-dimers, highly target-specific, and used under universal PCR conditions
  • Full primer coverage for Ion Torrent Ion AmpliSeq Exome Panel and Ion Torrent Ion AmpliSeq Cancer Hotspot Panel v.2 Sanger confirmation workflow
  • Flexible primer configuration to meet your research needs: primers can be ordered unmodified, M13-tailed, HPLC-purified, or desalted
  • All the primers have been checked by mass spectrometry and have passed stringent bioinformatics metrics. Lab bench validation test have shown >95% success rate

Expert recommendations for DNA primer design

Good primer design is essential for a successful PCR reaction. There are many factors to consider when designing the optimal primers for your gene of interest. Here are some tips to consider when designing primers.

Primer design tips

  • In general, a length of 18–30 nucleotides for primers is good.
  • Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.
  • If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
  • Aim for the GC content to be between 40% and 60%, with the 3' of a primer ending in C or G to promote binding.
  • Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting.
  • Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
  • Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
  • Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences).  These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
  • If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
  • If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
  • If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.

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Stylesheet for Classic Wide Template adjustments

For Research Use Only. Not for use in diagnostic procedures.