Custom oligos are an important tool that researchers can use in a wide variety of applications. Here are important design and ordering tips to get you to your experiments faster!

Aim for the GC content of your primer to be between 40% and 60% with the 3’ end of your DNA sequence ending in G or C to promote binding (known as a GC clamp). Since G and C bases have stronger hydrogen bonding than A and T, using them at the end of your sequence can help increase the stability of your primer. Be mindful, however, not to have too many repeating G or C bases in your sequence as this can cause primer-dimer formation.

The melting temperature (Tm) of the primers should be between 65°C and 75°C, and within 5°C of each other. Because the Tm is dependent on the length of the sequence, it’s important to keep primers minimal in length, if possible. The individual bases of the sequence also impact the Tm, with G and C bases resulting in higher melting temperatures than A and T bases. If the Tm of your primer is very low, try to find a sequence with more GC content, or slightly extend the length of the primer.

Specificity of the primer is usually dependent on both the sequence composition, length, and annealing temperature. An optimized length for primers is 18-30 bases. The shorter the primers are, the more efficiently they will bind or anneal to the target.

Additional tips for primer design are as follows:

• Restriction enzyme sites: Add three to four bases to the 5’ end of the sequence of your primer to allow for efficient digestion.
• Secondary structure: Use an even distribution of GC-rich and AT-rich domains to avoid secondary structure formations.
• Mononucleotides and dinucleotide repeats: Avoid runs of four or more of a single base or two base repeats (i.e., ACCCC, ATATATAT) which can cause primer mispriming events.
• Intra- or inter-primer homology: Avoid self-dimers and primer-dimers by eliminating both intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers that have complementary sequences).
• Cloning application: Purification is key—consider cartridge purification at a minimum.
• Mutagenesis application: Incorporate mismatched bases in the middle of the primer to introduce point-mutations into plasmid sequences.
• TOPO cloning application: DO NOT include a phosphate modification to ensure functionality with TOPO cloning kits.

When ordering your oligos, it’s important to know the difference between starting scale and final yield. Every processing step (i.e. base addition, purification, desalting) and modification addition is a chemical reaction that can diminish the yield of your oligo. When using the Thermo Fisher Oligo Design Tool, the minimum guaranteed amount based on sequence design and oligo options will be displayed. If the amount will not work for your experiments, consider using a larger synthesis scale or modifying your selected options/modifications.

While it’s true that purification options remove failure sequences or short-mers from the oligo sample to increase sample purity, this level of purification is not always necessary. The level of purity you need will depend on the application that you are using the oligo for, the length of the oligo, the desired final yield, and the modifications added to the oligo. For PCR or sequencing applications that don’t require ≥ 85% purity and use oligos that do not contain modifications, standard processing with ready-to-use-desalting options will often be the preferred choice. For applications like cloning, next-generation sequencing (NGS), or mutagenesis, or for oligos that contain modifications, high purity is critical and will require additional purification options using Cartridge, HPLC, or PAGE methods.

Ordering a custom oligo has never been easier through the use of our dynamic oligo design tool. When designing your oligo, the final yield and price will automatically update as you enter your sequence, synthesis scale, modifications, and purification options. Additional parameters are also displayed, including the minimum guaranteed yield, GC% of your sequence, and the melting temperature of your oligo. Once you have made your selections, simply add to your cart for checkout!

Using custom oligos in your research can get you to your results faster. We have optimized the design and ordering process to make your experience easy! For more information on custom oligo services, please see the links below.