QuikChange primer check

Stratagene's empirical Tm, plus forward/reverse pairing, mutation position and secondary structure.

Forward

—°C

Reverse

—°C

ΔTm

—°C

Paste a primer to start

Forward primer, 5′ → 3′

Type mutated bases in lowercase and the tool finds them. Labels and spacing are ignored.

Reverse primer, 5′ → 3′ — optional

Template, wild-type — optional, but makes the mutation checks exact

With a template, mismatches, insertions and deletions are found by alignment.

Substitution Insertion Deletion

Mismatched bases

Used only when there is no template and no lowercase.

Primer pair

—

Mutation

—

Design checks

—

Secondary structure

—

Nearest-neighbour Tm and oligo properties

—

Reaction conditions

Monovalent, mM

Mg²⁺, mM

dNTPs total, mM

Each primer, nM

Defaults approximate a QuikChange II reaction (Pfu buffer, 125 ng of a ~30-mer in 50 µl). Edit to match yours.

Benchling and IDT assume no Mg²⁺ by default, which is worth about 6 °C on a primer this length — use that preset when cross-checking their numbers, and the QuikChange one for what happens in the tube.

Stratagene Tm = 81.5 + 0.41(%GC) − 675/N − %mismatch, N = primer length, %mismatch = mismatched bases ÷ N × 100. For insertions and deletions the mismatch term is dropped and N excludes the inserted bases — a deletion primer doesn't contain the deleted bases, so its N is simply its length. The 78 °C target is a design heuristic for this formula only; it is not an annealing temperature and will not agree with NEB or IDT.

Nearest-neighbour Tm uses SantaLucia (1998) unified parameters with von Ahsen's Mg²⁺ correction, for each primer against its perfect complement. That is the Tm once the mutation is copied in; mismatches against the original template lower the first cycles.

Hairpins and self-dimers are scored as Watson–Crick stems only (no mismatches, bulges or dangling ends), ΔG at 37 °C. Flags follow the usual IDT-style rule of thumb: hairpins below −3 kcal/mol (−2 if the 3′ end is in the stem), dimers below −9 kcal/mol (−5 if a 3′ end is paired). Treat these as a screen, not a prediction — and note that a standard QuikChange pair is a perfect hetero-dimer on purpose, which is why that number is only shown for designs where the two primers are not full-length complements.