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Site-Directed Mutagenesis Primer Designer

Design mutagenic primers for Q5 (back-to-back) or QuikChange (overlapping) site-directed mutagenesis. Point substitution, insertion, deletion, and block substitution. Mismatch-aware annealing Tm, hairpin and GC warnings, CSV export. All computation runs client-side.

Q5 / QuikChange SDMMismatch-Aware TmClient-Side

Try it out

Load example Site-Directed Mutagenesis Primer Designer data to see the full workflow

Template Sequence

Paste your plasmid or template DNA sequence (ACGT only, whitespace and digits ignored).

Mutation Settings

Change a single base

NEB Q5 SDM Kit style — non-overlapping primers, mutation on forward only

Position in template where the mutation starts

  • Design mutagenic primers for point substitutions, insertions, or deletions
  • Choose between Q5 (back-to-back) and QuikChange (overlapping) primer strategies
  • Calculate mismatch-aware annealing temperatures
  • Check primers for hairpins, homopolymer runs, and GC clamp issues
  • Export primer sequences as CSV for ordering

Don't use for

  • Large insertions (>50 bp) — consider Gibson Assembly or restriction cloning
  • Whole-gene synthesis — use a gene synthesis service
  • Random mutagenesis — use error-prone PCR or chemical mutagenesis
  • CRISPR-based editing — use a guide RNA design tool instead

How site-directed mutagenesis works

Site-directed mutagenesis introduces targeted changes into plasmid DNA using mutagenic oligonucleotide primers.

Q5 / back-to-back method (NEB): 1. Two non-overlapping primers bind back-to-back on the template 2. Only the forward primer contains the mutation 3. Exponential amplification produces the mutant plasmid 4. KLD (kinase-ligase-DpnI) treatment circularizes and selects for mutant DNA
QuikChange / overlapping method: 1. Two complementary primers both carry the mutation 2. Linear amplification extends both primers around the plasmid 3. DpnI digests methylated (wild-type) template 4. Nicked circular mutant DNA transforms into E. coli for repair

Rules for mutagenic primer design

Good mutagenic primers follow these guidelines:

  • Tm of binding region: 60-65 °C (using nearest-neighbour calculation)
  • GC content: 40-60% across the full primer
  • 3′ GC clamp: End with G or C for stable template binding
  • No hairpins: Avoid self-complementary regions >4 bp
  • No homopolymer runs: Avoid 4+ identical consecutive bases
  • Primer length: Typically 25-45 nt for the full primer including mutation
  • Mismatch position: For Q5, place the mutation at or near the 5′ end of the forward primer
  • For insertions: Added bases go at the 5′ end of the forward primer (Q5) or centered (QuikChange)

Troubleshooting SDM experiments

Common issues and solutions:

  • No colonies: Check primer Tm (use annealing Tm, not full Tm), verify DpnI digestion, ensure template is dam-methylated
  • Wild-type background: Increase DpnI digestion time, use more DpnI, verify dam+ host strain
  • Deletions/rearrangements: Reduce extension time, use high-fidelity polymerase (Q5), lower template amount
  • Low efficiency with insertions >6 bp: Consider two-step cloning or Gibson Assembly instead
  • Multiple mutations needed: Introduce one at a time or use Q5 Multi SDM Kit for 2-5 simultaneous changes

Frequently Asked Questions

Next Steps

PCR & qPCR Instruments

Thermal cyclers for SDM amplification and colony screening PCR

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Gel Electrophoresis Systems

Verify PCR amplification and screen mutagenized constructs

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