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Primer design puzzle

Tune two primers to amplify the target region so both melt near the target Tm and match each other — but the two ends have different GC, so you can't tune them the same way. Graded by the real thermodynamics engine.

mode: primer design · cloning · directed evolution

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Position 5'→3' along the top strand. The lit band ▮ between the primers is the amplicon — the sequence that gets copied. ▮ forward primer (top strand, left end)   ▮ reverse primer (reverse-complement of the right end, primes the bottom strand)  ·  greener shading = GC-richer.

Forward primer · 5'→3'

len 20 nt

Reverse primer · 5'→3'

len 20 nt
melting temperature — where the two primers sit, and the gap you'd have to bridge with one annealing temp
Adjust the primer lengths, then run PCR.