How long should the flanking regions be?

10–15 bp of perfect template match on each side of the mutation is usually enough, giving primers of roughly 25–45 nt. Longer flanks raise the annealing temperature and cost more.

Why is DpnI digestion needed?

The parental plasmid was propagated in E. coli and is methylated; the PCR product is not. DpnI digests only the methylated template, removing the wild-type background.

Can I make deletions with this tool?

Yes. Set the range you want removed and leave the replacement empty; the length change will show as a negative number.

What about insertions?

Set start = end + 1 so the range is empty, then type the sequence to insert. The product grows by exactly that many bases.

Site-Directed Mutagenesis Primer Designer

Design a back-to-back primer pair for point mutations, insertions and deletions — Q5 style, with the product sequence.

Template sequence

0 bp

Start (1-based)

End (1-based)

Replacement

Flank (bp)

Coordinates are 1-based and inclusive. Use start = end + 1 for a pure insertion, or an empty replacement for a deletion.

Paste a template sequence to design the primer pair.

Description

Paste your template, give the 1-based range to replace and the replacement sequence. The tool returns the mutated product and a primer pair that carries the change at its 5′ end and anneals to the template on both sides.

How to use

Set start and end to the bases you want replaced (1-based, inclusive). For a point mutation, set start = end and type the new base. For an insertion, set start = end + 1. For a deletion, leave the replacement empty.

Learn more

What it does

Site-directed mutagenesis introduces a defined change into a plasmid by amplifying the whole template with a pair of back-to-back primers, one of which carries the mutation. After DpnI digestion of the methylated parental template and blunt-end ligation, the product is a closed mutant plasmid.

How it works

The forward primer is built from the template bases immediately upstream of the target, followed by the replacement, followed by the bases immediately downstream — the flanking length you choose on each side. The reverse primer is its reverse complement, so the two amplify the entire plasmid in opposite directions. The reported product is the template with the target range substituted.

Worked example

To change base 12 of a 300 bp template from G to A, set start = end = 12 and replacement = A with a 20 bp flank. The forward primer is the 20 bases before position 12, then A, then the 20 bases after — 41 nt, which is a typical SDM primer length.

When to use it

Use it for single-residue substitutions, small insertions such as a tag or a restriction site, and clean deletions. For changes larger than about 25 bp, consider splitting the change across two primers or switching to a two-fragment assembly.

FAQ

How long should the flanking regions be?: 10–15 bp of perfect template match on each side of the mutation is usually enough, giving primers of roughly 25–45 nt. Longer flanks raise the annealing temperature and cost more.
Why is DpnI digestion needed?: The parental plasmid was propagated in E. coli and is methylated; the PCR product is not. DpnI digests only the methylated template, removing the wild-type background.
Can I make deletions with this tool?: Yes. Set the range you want removed and leave the replacement empty; the length change will show as a negative number.
What about insertions?: Set start = end + 1 so the range is empty, then type the sequence to insert. The product grows by exactly that many bases.

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