How to Design PCR Primers: Designing, Checking & Ordering Primers
Designing primers is one of the most fundamental steps in molecular biology. Whether you’re setting up a diagnostic PCR, cloning a gene, or preparing for qPCR, getting your primers right can mean the difference between clean, specific amplification and frustrating, non-specific bands.
In this post, we’ll walk through the complete primer design workflow — from sequence retrieval, to checking primer quality, to ordering them from a synthesis company.
Step 1. Retrieve Your Target Sequence
The first step is to obtain the DNA sequence you want to amplify:
For genomic DNA, you can download sequences directly from databases such as NCBI.
For organism-specific projects, use specialized databases (e.g. PlasmoDB for Plasmodium).
For cloning, always use the cDNA sequence (introns removed). Genomic DNA may be fine for general PCR, but introns will complicate cloning.
Download sequences in FASTA format (beginning with “>” followed by the header, then nucleotide sequence). Store it in a Word doc or text editor for easy manipulation.
Step 2. Choose a Primer Design Tool
Manually designing primers is possible, but software saves time and reduces error. Popular tools include:
Benchling, SnapGene (free access)
Geneious (subscription)
These tools will identify unique forward and reverse primers at the right positions on your target gene.
Step 3. Check for Secondary Structures
Good primers should not fold back or bind to each other. Test for:
Hairpins – intramolecular folding that prevents proper binding.
Primer dimers – primers binding to each other rather than the target (can be self-dimers or cross-dimers).
If secondary structures are predicted, discard those primers and choose alternatives.
Step 4. Check Specificity with BLAST
Once you’ve shortlisted primers, confirm they don’t bind elsewhere in the genome:
Run them through NCBI BLAST.
Look for off-target alignments across other regions of the genome.
Accept only primers with very low expectation (E) values at unintended sites.
Small mismatches near the 3′ end are especially important — if mismatches exist here, off-target amplification is unlikely.
Step 5. Verify Binding on the Original Sequence
Paste your primers back into the original sequence and confirm:
Forward primer should match directly.
Reverse primer will need to be checked using a reverse complement tool.
This step ensures you know exactly where your primers bind on the gene.
Step 6. Check Melting Temperatures (Tm)
Primer performance depends heavily on temperature. Keep in mind:
Typical primer length: 18–25 bases
GC content: 40–60%
Tm: 55–65 °C
Melting temperature requirements of the forward and reverse primers should be within 5 °C of each other
Always adjust primer length to achieve the correct Tm for your chosen DNA polymerase.
Step 7. Order Your Primers
Once you’re satisfied, it’s time to order from an oligo synthesis company. Most suppliers provide an Excel order form to fill in. You’ll need to specify:
Purification method:
Standard (desalted)
High purity (HPLC, PAGE, cartridge) — higher cost, higher quality
Synthesis scale: (e.g. 25 nmol, 50 nmol). Note: this is an amount, not a concentration.
Modifications (if required):
For standard PCR: no modifications needed
For qPCR: add fluorescent probes (FAM, HEX, etc.)
Fill in the spreadsheet, submit your order, and wait for delivery.
Step 8. Resuspend and Use
When primers arrive, resuspend them to a working concentration (usually 100uM) and store them in aliquots. 10uM aliquots are usually made and stored in low-binding tubes at -20oC. They are now ready to use in your PCR reactions.
Conclusion
Designing primers may sound complex, but following this workflow ensures that your primers are:
Specific (bind only to your target)
Stable (no dimers or hairpins)
Efficient (correct Tm for amplification)
By combining bioinformatics tools with careful checks, you can go from sequence to PCR-ready primers with confidence.
🎥 Watch my full video tutorial where I walk through this process step-by-step — from downloading sequences to filling in the synthesis company Excel form.
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