Choose the experiment type first: PCR primer validation, qPCR primer and probe checks, large oligo pool QC, or CRISPR sgRNA library preparation. Each guide shows which checks matter before ordering and which calculator to open when you need a specific result.
| Use Case | Start With | Decision Signals | When to Open a Tool |
|---|---|---|---|
| Validate PCR Primers | PCR primer checklist | suitable for synthesis, needs optimization, or should be redesigned | Open Tm Calculator only for the Tm step |
| Validate qPCR Primers and Probes | qPCR assay checklist | chemistry choice, probe Tm, dimer risk, or multiplex readiness | Open Primer Analyzer for qPCR primer review |
| QC a Large Oligo Pool | Oligo pool QC guide | batch passes, needs filtering, or needs vendor review | Open Batch QC for sequence upload |
| Design a CRISPR sgRNA Library | CRISPR sgRNA library guide | enough guides, clean sequences, and synthesis-ready pool | Open Coverage Calculator for library sizing |
After this page narrows the job, open the matching guide or calculator: Tm Calculator, Primer Analyzer, Batch Sequence QC, or Oligo Pool Design Guide.
Use this comparison before you request quotes. For pooled libraries, confirm current vendor capability, pricing, delivery format, and QC scope before treating a design as order-ready.
| Planning Scenario | Best For | Confirm Before Quote | QC Risk | Related Guide |
|---|---|---|---|---|
| Large pooled library | CRISPR screens, capture panels, and variant libraries | Pool-size tier, full oligo length, delivery amount, and QC report format | Representation and dropout should be verified with NGS when the experiment depends on broad coverage. | Oligo pool design guide |
| Small or focused sequence set | PCR primers, validation oligos, focused panels, and low-count pools | Scale, purification, modifications, naming rules, and accepted bases | Individual sequence quality may matter more than pool-wide uniformity. | Format adapter |
| Specialized or quote-dependent pool | Long constructs, modified oligos, unusual bases, or constrained delivery formats | Feasibility review, failure policy, QC scope, and whether redesign is required before synthesis | Treat vendor feedback as part of the design loop, not just an order step. | Vendor comparison |
Note: Vendor specifications change. Check the vendor's current documentation before relying on sequence limits, pool-size tiers, delivery amounts, or QC report formats for a live order.
Choose SYBR or probe chemistry, align primer and probe Tm, and review dimer or probe risks before synthesis.
Relevant checks
Decide whether a primer pair is suitable for synthesis, needs optimization, or should be redesigned.
Relevant checks
Run batch sequence QC, estimate synthesis risk, and review pool uniformity before vendor submission.
Relevant checks
Size your library, screen guides for GC and sequence issues, and prepare a cleaner oligo-pool submission.
Relevant checks
Oligonucleotide projects carry different risks depending on how the sequence will be used. A primer pair needs paired Tm, GC balance, specificity, and dimer checks. A pooled library needs sequence-format validation, length consistency, composition outlier review, and synthesis-risk flags. A CRISPR screen also needs enough guides per target and enough library representation after selection.
Start with the guide that matches the experiment rather than opening a calculator at random. Use the Tm Calculator when melting temperature is the next decision, the GC Analyzer when composition is the limiting factor, Batch Sequence QC when a sequence set needs screening, and the Coverage Calculator when library size is the main question.
Primer validation, oligo pool QC, and CRISPR library preparation use overlapping measurements, but the pass/fail decision is not the same. A primer pair may tolerate a narrow optimization step; a pooled library may fail because a small fraction of sequences are hard to synthesize or underrepresented. Keeping each guide focused makes the checklist easier to follow before ordering.
For formulas and scientific background, use the reference pages. For a concrete calculation, open the calculator. For an ordering decision, start with the relevant use-case guide and confirm the current vendor requirements before submission.
Choose this guide when you already have forward and reverse primer candidates and need a synthesis decision. Check paired Tm, GC content, amplicon context, and structure risk before deciding whether to proceed, optimize, or redesign the pair.
Use this guide when: the next decision is whether the primer pair is suitable for synthesis, needs gradient optimization, or should be redesigned.
Guide: PCR Primer Validation Checklist Before Ordering. Open the calculator pages from there when a specific measurement is needed.
Large oligo pools require systematic QC: sequence validation, length-window review, Tm uniformity, GC distribution, and error-risk screening before vendor submission. This guide keeps the checks in a practical order and leaves current vendor specifications to vendor documentation and vendor confirmation. The recommended check combines Batch Sequence QC (FASTA upload, automated validation), Pool Uniformity Estimator (CV calculation for Tm, GC%), and Error Rate Calculator (composition-based error prediction). Also use Tm Calculator to verify median Tm matches experimental conditions.
QC metrics: (1) Sequence diversity—check for duplicates, excessive homology (>85% identity), homopolymer runs (>6 nt); (2) Tm uniformity—calculate Tm for all sequences, target CV < 10% (strict hybridization) or < 20% (amplification); (3) GC distribution—median 45-55%, avoid extreme outliers (<30% or >70%); (4) Synthesis compatibility—flag sequences with prohibitive motifs (e.g., GGGG runs for some platforms). For NGS library prep, see How to Run Pre-Order Oligo Pool QC.
Suggested check (45-90 min): Upload pool sequences to Batch QC, export Tm/GC data, analyze in Uniformity Estimator, estimate error rates per Error Calculator. Filter outliers iteratively until QC thresholds met. Essential for multiplexed assays, variant libraries, and CRISPR libraries.
CRISPR sgRNA library design (with SpCas9 as the default example) requires coverage optimization (genome-wide: 3-10 guides/gene, ~50,000-100,000 total; focused: 10-20 guides/target), sequence specificity (minimize off-targets with >2 mismatches), guide activity prediction (GC content 40-60%, avoid TTTT poly-T terminator), and synthesis constraints (20 nt guide for SpCas9; note: SaCas9 uses 21-23 nt, Cas12a uses 23-25 nt). The guide uses Coverage Calculator (library size vs. target complexity), Batch Sequence QC (sequence validation, duplicate detection), GC Analyzer (activity prediction), and Secondary Structure Predictor (guide RNA folding).
Design criteria: (1) Coverage—genome-wide screens need 3-10 guides per gene for statistical power; focused libraries can use 10-20 guides for critical targets. Calculate library size requirements in Coverage Calculator. (2) Guide activity—target GC 40-60%, avoid TTTT (Pol III terminator), position guides early in CDS for knockout efficiency. (3) Specificity—check off-target potential using specialized tools; prioritize guides with >3 mismatches to other genomic sites. (4) Synthesis—standard format is 20 nt guide + constant scaffold (~80 nt total); use Oligo Pool QC for final validation.
Suggested check (60-120 min): Calculate library size in Coverage Calculator, design guides using external tools (e.g., CRISPRDesign databases), import sequences to Batch QC, filter by GC% in GC Analyzer, check scaffold folding in Structure Predictor. Advanced users should understand statistical power requirements and screening methodology. See Scientific References for CRISPR design publications.
For SantaLucia, Owczarzy, Breslauer, and other method references behind the calculators, visit the Scientific References page.
For primer-specific troubleshooting, use the PCR primer checklist. For pool-order decisions, use the oligo pool QC guide. For CRISPR library sizing, use the CRISPR sgRNA library guide.
Continue with the guide that matches the next decision. Open references when you need method background.
Use this when a forward and reverse primer pair needs a proceed, optimize, or redesign decision.
Use this when a pooled sequence set needs sequence, uniformity, and synthesis-risk review.
Use this when guide count, library coverage, and pool submission quality are the decision.
Use this for SantaLucia, Owczarzy, and the primary methods behind the calculators and QC thresholds.
Start with the guide that matches your experiment, then open calculators or reference pages when a specific check is needed. Need help with a specific application? to request a custom use case guide.
Can't find a guide for your specific application? Let us know what you're working on, and we may create a custom use case guide for you.