Annealing temperature, GC clamps and why a 5°C Tm mismatch kills your reaction — design primers that amplify.
Estimate the melting temperature (Tm) of a pcr primer — Wallace rule for ≤13 nt, basic GC formula for longer oligos. Balance forward/reverse primer Tm within ~5°C.
Estimate the melting temperature (Tm) of a qpcr primer — Wallace rule for ≤13 nt, basic GC formula for longer oligos. Balance forward/reverse primer Tm within ~5°C.
Estimate the melting temperature (Tm) of a cloning primer — Wallace rule for ≤13 nt, basic GC formula for longer oligos. Balance forward/reverse primer Tm within ~5°C.
Calculate the GC content (%) of primer quality (aim 40–60%). GC% drives melting temperature, secondary structure and PCR behaviour.
Calculate the GC content (%) of an expected amplicon. GC% drives melting temperature, secondary structure and PCR behaviour.
Calculate the GC content (%) of a plasmid backbone. GC% drives melting temperature, secondary structure and PCR behaviour.
Generate the reverse complement (5′→3′) of a PCR Primer sequence — for designing the reverse primer. Runs in your browser; the sequence is never uploaded.
Calculate the GC content (%) of genomic or any DNA. GC% drives melting temperature, secondary structure and PCR behaviour.
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