Oligo Design Methods and Workflow Paths
Choose the next method page, calculator, or workflow for primer design, Tm decisions, CRISPR libraries, oligo pool QC, and synthesis planning.
Core Workflow Paths
Design & Validate PCR Primers
Choose primer length, check Tm and GC, screen dimers, and troubleshoot failed PCR before ordering.
Plan Oligo Pool Synthesis
Move from pool design and synthesis method choice to vendor selection, QC expectations, and order readiness.
Why Do Tm Calculators Disagree?
See why NEB, IDT, and Primer3 disagree, how salt and Mg2+ shift Tm, and which number to trust for PCR or qPCR.
Design & QC a CRISPR sgRNA Library
Choose the right library format, rank sgRNAs, size coverage, define oligo architecture, and review QC before screening.
Focused Method Decisions
Which Tm Calculation Method to Use?
Decision tree for choosing between SantaLucia NN, Owczarzy salt correction, Wallace rule, and %GC methods.
Resolving Tm Discrepancies Between Tools
Why different tools report different Tm values and how to reconcile discrepancies.
Diagnose PCR Failure from Hairpins & Primer Dimers
Use gel clues, ΔG thresholds, and tool checks to decide whether hairpins, self-dimers, or cross-dimers are blocking amplification.
What QC Should I Request for Oligos?
Decide what vendor QC to request, how to read QC reports, and how to review oligo pool quality before and after synthesis.
Oligo Pool Error Rates & Coverage
Understanding synthesis error rates, dropout rates, and how to calculate required sequencing depth.
Design Illumina Adapters & Dual Indexes
Choose adapter structure, set up UDI, prevent index hopping, and QC custom Illumina-compatible adapters before ordering.
Primer3 Thermodynamic Penalty Scoring
How Primer3 evaluates primer quality using thermodynamic penalty scores, and how to interpret the results.
Method References
Understanding GC Content
What GC content means for your oligos, why it matters for synthesis and PCR, and how to interpret GC distribution.
IntermediateDNA Secondary Structures Explained
Hairpins, self-dimers, G-quadruplexes, and i-motifs. How they form, why they matter, and how to predict them.
IntermediateOligonucleotide Synthesis Methods
Column-based vs array-based synthesis. Phosphoramidite chemistry explained. How synthesis method affects quality.
Oligo Pool Workflows
Move from pool architecture to synthesis choice, vendor short-listing, QC checks, and application-specific execution.
Solutions by Audience
For Academic Labs
Free tools with no login — built for grad students, postdocs, and PIs.
For Biotech Companies
Scale from target discovery to assay development with zero IT overhead.
For Pharma R&D
Pharma-grade data privacy. Reproducible calculations for documentation.
Tool Comparisons
Head-to-head comparisons of popular oligo analysis tools and synthesis vendors.
OligoPool vs IDT OligoAnalyzer
Full feature comparison matrix
OligoPool vs NEB Tm Calculator
Same algorithm, ±0.3°C accuracy
Best Free Oligo Tools 2026
Comprehensive annual roundup
Tm Methods Compared
5 methods, interactive tool
Top 5 Primer Tools
Ranked with pros & cons