Site-Directed Mutagenesis Primer Designer (QuikChange & Q5)
Design SDM primers from a nucleotide or amino-acid change, QuikChange or Q5 style.
🔒 Local processing — pasted sequences are not uploaded
Design site-directed mutagenesis (SDM) primers without hand-counting bases. Paste your template and specify the change either at the nucleotide level (a point mutation by position) or the amino-acid level — pick the residue and the target amino acid and the tool chooses a codon with the fewest changes. It returns a QuikChange-style overlapping primer pair or Q5/NEBaseChanger-style back-to-back primers, with nearest-neighbour binding-arm Tm, a recommended annealing temperature, and the full mutated sequence and protein.
0 bp
Residue out of range
QuikChange-style primers are fully complementary and centred on the change; Q5/NEBaseChanger-style primers sit back-to-back (the change at the 5′ end of the forward primer) and amplify the whole template. Binding-arm Tm uses SantaLucia nearest-neighbour thermodynamics. The recommended annealing temperature is a guide — follow your polymerase’s protocol.
How to use the Mutagenesis Primers tool
- 1Paste the template and choose an amino-acid or nucleotide edit.
- 2Set the residue + target amino acid (or position + new base), and pick QuikChange or Q5 primer style.
- 3Copy the mutagenic primer pair (mutation highlighted) and note the recommended annealing temperature.
Frequently asked questions
- How does it choose the mutant codon for an amino-acid change?
- Among all codons for the target amino acid, it picks the one that differs from the original codon in the fewest positions (to keep the primer mismatch small), breaking ties by codon-usage frequency in the organism you select.
- What is the difference between QuikChange and Q5 primers?
- QuikChange-style primers are fully complementary and centred on the mutation (used with whole-plasmid linear amplification and DpnI digestion). Q5/NEBaseChanger-style primers sit back-to-back with the change at the 5′ end of the forward primer and are ligated after PCR. The tool builds either from the same edit.
- How is the annealing temperature determined?
- From the nearest-neighbour Tm of the template-binding arms (SantaLucia 1998); the recommended value is roughly 3 °C below the limiting arm. Treat it as a starting point and follow your polymerase's protocol.