Primer Design Tips & Tools
Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest. Here are some tips to consider when designing primers.
- 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 TOPO® cloning, the primers should not have a phosphate modification.
Designing & Ordering Primers With Vector NTI Advance® Sequence Analysis Software
Designing and ordering primers takes about 60 seconds when you use Vector NTI Advance® software:
- Select the region to amplify.
- Access the primer design menu and select “amplify selection”.
- Add 5' and 3' restriction sites, if required, to the sense and antisense primer.
- Add additional bases, if required, to the sense and antisense primer.
- Inspect the left pane for thermodynamic parameters such as Tm, length, and GC content.
- Select the product, and click “order”.
- Add a researcher name, any desired 5' or 3' modifications, or change the purity.
- Click “add to cart” and begin the checkout process.
Visit the Vector NTI Advance® Sequence Analysis Software page for more information and to view a video that demonstrates the simple steps of designing and ordering the right primers.
OligoPerfect™ Designer is a free, simple, and efficient Web-based primer design tool that works with a DNA template sequence you upload. Like Vector NTI Advance® software, OligoPerfect™ Designer is seamlessly connected to our online ordering system, so you never have to cut and paste sequences.
For Research Use Only. Not for use in diagnostic procedures.