OligoPool calculators use published thermodynamic models, visible assumptions, and the shared browser-default public-tool boundary. This page explains which scientific methods support each tool, where the limits are, and which page to use for the actual calculation.
Use this methodology page when you need algorithm transparency before trusting a result. Use the Scientific References page for complete citations and the Accuracy Validation page for validation data.
Methodology Principles
Use published scientific models first, not proprietary black-box claims.
Keep calculation assumptions visible on the page where the user enters data.
Separate direct calculation intent from explanation, comparison, and reference pages.
Keep public sequence calculations browser-default, with explicit save and API paths documented separately.
Disclose limitations so users know when empirical validation or vendor guidance is still required.
Primer and oligo melting temperature is calculated with nearest-neighbor thermodynamics, salt correction, oligo concentration, and optional solvent adjustments.
Unmodified DNA/RNA is supported directly. Modified bases, LNA, PTO, 2'-O-methyl RNA, and labeled probes need empirical adjustment or vendor-specific parameters.
GC Content and Composition
GC content uses direct base composition, sliding-window review, and threshold interpretation for primer, adapter, guide, probe, and pool screening.
Zuker / mfold and UNAFold-style folding literature
PCR primer dimer threshold practice
Known limitations
Short-oligo structure scoring is a screening method. Experimental PCR failures can also come from template complexity, off-target binding, polymerase choice, or cycling conditions.
Batch Sequence QC
Pool-scale QC combines sequence length, GC range, homopolymer runs, low complexity, Tm outliers, and structure-risk flags before synthesis or vendor upload.
Batch QC reduces preventable design risk before ordering, but it does not replace vendor-side synthesis QC, NGS validation, or application-specific wet-lab testing.
Mass, Extinction, and Dilution
Molecular weight, extinction coefficient, concentration, and dilution calculations use standard nucleotide mass and Beer-Lambert relationships.
Coverage and uniformity estimates are planning aids. Final performance depends on synthesis platform, amplification bias, sequencing depth, and experimental design.
Paper, DOI, PMID, method role, and implementation relevance.
Privacy as a Methodology Choice
Many oligo and primer sequences are unpublished assay designs, CRISPR libraries, diagnostic probes, or customer-specific research materials. For that reason, OligoPool calculators are designed around browser-default computation where practical. Calculations run in your browser by default. Dashboard History can store the full input snapshot when auto-save, session, or account history is enabled; Library saves and membership/API workflows can send sequence assets by design.
See the Privacy Policy for the site-level data statement, and use browser developer tools if you need to inspect network behavior during sensitive analysis.