Primer Design Tool
Design PCR primer pairs from a template and a target region, ranked by Tm match and GC content.
Template length: 0 nt
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What it does
Designing a primer pair by eye means balancing four things at once: the annealing temperature of both primers, their GC content, the length of the product, and where the 5' ends land. This tool does that search for you. Give it a template sequence and the region you want to amplify, and it returns ranked primer pairs you can copy straight into an order form.
How it works
The search scans both strands for candidate primers of 18–25 nt whose 3' end sits within 60 bp of your target boundary — the forward primer upstream of the target start, the reverse primer downstream of the target end. Each candidate is filtered by GC content (40–60%) and by how close its melting temperature is to 60 °C, then scored on the Tm deviation, the GC content, and how many G/C bases sit in the last five nucleotides. The 3' GC clamp scoring favours one or two G/C bases there: none makes the 3' end too weak to initiate, while an all-G/C clamp tends to misprime. Finally every forward/reverse combination is scored together and the ten best pairs are returned.
Worked example
Load the sample — a 750 bp template with the target region set to 121–420. The top-ranked pair is 5'-CACGCACCTAACAGTCTAAG-3' and 5'-GTAACTCGCTAGGTTGGGTA-3'. Both are 20 nt long with a Tm of 59.5 °C and 50% GC, and they amplify a 343 bp product. The forward primer binds at position 77 and the reverse at position 400, so the amplicon spans the target with room on both sides for the polymerase to load.
When to use it
Use it to plan a colony PCR screen around an insert, to amplify a region for cloning where you need matching Tm values, or to rescue a reaction that failed because the original primers had mismatched annealing temperatures. Once you have a pair, run it through the primer checker to rule out hairpins and 3' dimers before ordering.
FAQ
- How does this primer design tool choose a primer pair?
- It scans candidate primers of 18–25 nt on both strands around your target region, keeps only those with a GC content of 40–60% and a Tm within 8 °C of 60 °C, then scores each pair on Tm match, GC content and the 3' GC clamp. Pairs are ranked by total score and the best ten are shown.
- Why does the tool return no primer pairs for my target?
- The most common cause is a target sitting too close to the 5' end — the forward primer needs roughly 60 bp of upstream space. Very short targets can also fail because no pair fits the product window. Move the target toward the middle of the sequence or widen it.
- What melting temperature does the tool target?
- 60 °C, with a tolerance of ±8 °C. That is the usual starting point for a two-step PCR and keeps the forward and reverse primers compatible with a single annealing temperature. Verify individual primers with the Tm calculator if you need tighter matching.
- Are the designed primers checked for hairpins or dimers?
- Not during design. Copy the pair into the primer checker to test both primers for hairpins, self-dimers and cross-dimers, and to scan the template for off-target binding sites.