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Q5 / QuikChange SDMFree in-browser calculator

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.

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Validated2026-04-07
CitableMethods and citation included

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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

When to use

  • 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

Do not 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

Use annealing Tm, not full Tm

The full primer Tm includes mismatched bases. For setting PCR annealing temperature, use the annealing Tm (matched bases only) shown in the results. This prevents failed amplification from over-estimated Tm.

Q5 method for insertions and deletions

The back-to-back Q5 method handles insertions and deletions more reliably than QuikChange. For point substitutions, both methods work well.

DpnI step is critical

DpnI digests dam-methylated template DNA but not the PCR-amplified mutant. If your template is not dam-methylated (grown in dam- strain), DpnI selection will not work.

Verify by sequencing

Always sequence the entire mutagenized region. PCR can introduce secondary mutations, even with high-fidelity polymerases. Send 2-3 clones for sequencing.

Template concentration matters

Use 1-25 ng of template for Q5 SDM. Too much template increases wild-type background; too little reduces colony count.

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Method

Primer design follows NEB Q5 SDM Kit guidelines (back-to-back) and Zheng et al. (2004) QuikChange protocol (overlapping). Tm calculation: SantaLucia (1998) nearest-neighbour with salt correction. Annealing Tm uses only the perfectly matched binding region. Secondary structure checks: hairpin stem detection (4+ bp), homopolymer run detection (4+ consecutive identical bases), GC clamp analysis.

2

Validated

Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

3

How to cite

How to Cite

ConductScience Site-Directed Mutagenesis Primer Designer (v1.20.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/site-directed-mutagenesis-primer-designer

Liu H, Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008;8:91. doi:10.1186/1472-6750-8-91

Zheng L, Baumann U, Reymond JL. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004;32:e115. doi:10.1093/nar/gnh110

SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. PNAS. 1998;95:1460–1465. doi:10.1073/pnas.95.4.1460

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

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