Oligonucleotide Design & Tm Calculator FAQ

Q: What is an oligo pool (oligonucleotide pool)?

What is an oligo pool (oligonucleotide pool)? - See detailed answer on page

Q: What is oligonucleotide design and why does it matter?

What is oligonucleotide design and why does it matter? - See detailed answer on page

Q: How do I choose the right tm calculator for my primers?

How do I choose the right tm calculator for my primers? - See detailed answer on page

Q: What method is used for Tm calculation?

What method is used for Tm calculation? - See detailed answer on page

Expert answers on tm calculator accuracy, oligonucleotide design principles, primer secondary structure analysis, oligo pool synthesis, and molecular weight calculations. Get science-backed guidance for PCR primer design, GC content optimization, and batch sequence analysis. Can't find your answer? Check our User Guide for detailed documentation, or explore use case examples for step-by-step workflows.

Oligonucleotide Basics

Q:What is an oligo pool (oligonucleotide pool)?

An oligo pool is a complex mixture of thousands to millions of unique oligonucleotides synthesized in a single reaction. Each oligo typically ranges from 40-300 nucleotides. Oligo pools are manufactured using array-based synthesis (silicon chips) or microfluidic platforms, then cleaved and pooled together.

Applications: CRISPR gRNA libraries, MPRA regulatory element screening, DNA data storage, antibody display libraries, synthetic gene assembly, variant libraries, and high-throughput functional genomics.

Key Quality Metrics (as of 2025):

Vendor	Technology	Error Rate	Pool Size
Twist Bioscience	Silicon array	~1:3,000	Up to 1M+ sequences
Agilent	SurePrint array	~1:500-1:1,500	Up to 244K sequences
IDT (oPools)	Array-based	~1:1,500	Up to 100K sequences
CustomArray	Array synthesis	~1:200-1:500	Up to 12K sequences

Source: Vendor specifications as of November 2025. Twist Bioscience error rate verified from official FAQ. Error rates vary by oligo length and complexity.

Q:What is oligonucleotide design and why does it matter?

Oligonucleotide design is the process of creating DNA or RNA sequences optimized for specific applications: PCR primers, qPCR probes, sequencing adapters, CRISPR guide RNAs, or synthetic genes. Critical design parameters include: 1) Melting temperature (Tm): Controls binding specificity and PCR annealing temperature, 2) GC content: Affects stability (target 40-60% for primers), 3) Secondary structure: Hairpins and dimers reduce efficiency, 4) Specificity: Off-target binding causes false amplification. Poor design leads to failed experiments, wasted reagents, and incorrect results. Use our Tm Calculator and Secondary Structure Predictor to validate your designs.

Q:How do I choose the right tm calculator for my primers?

Tm calculator choice depends on oligo type and application: 1) PCR primers (15-30nt): Use nearest-neighbor methods (SantaLucia 1998) like our Tm Calculator - accurate within ±2°C, 2) Long oligos (>50nt): Salt-adjusted nearest-neighbor with unified parameters, 3) Modified oligos: Check if calculator supports LNA, PNA, or other modifications. Avoid simple GC-based formulas (Wallace rule) for primers >14nt - they're inaccurate (±5°C error). Key features: salt correction (Na⁺, Mg²⁺, dNTPs), concentration adjustment, and published validation. Compare your calculator results with experimental data for your specific buffer conditions.

Q:What is primer secondary structure and how does it affect PCR?

Primer secondary structure refers to intramolecular (self-folding) or intermolecular (dimer) interactions that compete with target binding. Three critical types: 1) Hairpins: Stem-loop structures within a single primer (ΔG < -3 kcal/mol problematic), 2) Self-dimers: Two copies of the same primer binding together, 3) Hetero-dimers: Forward and reverse primers binding to each other. Impact on PCR: reduced primer availability, primer-dimer artifacts (visible as ~40-100bp bands on gels), lower target amplification efficiency, and false positives in qPCR. Prevention: Keep ΔG > -5 kcal/mol for 3' end interactions, avoid complementary 3' ends between primer pairs, and verify with Secondary Structure Predictor before ordering.

Q:What are the key oligonucleotide properties I need to calculate?

Essential oligo properties for molecular biology applications: 1) Melting Temperature (Tm): Predicts annealing temperature for PCR/hybridization (see Tm Calculator), 2) GC Content: Affects stability and specificity - target 40-60% for primers (GC Analyzer), 3) Molecular Weight: Required for accurate resuspension to desired concentration (MW Calculator), 4) Extinction Coefficient (ε260): Used to calculate concentration from A260 measurements, 5) Secondary Structure (ΔG): Predicts hairpins and dimers (Structure Predictor). Advanced: Nearest-neighbor thermodynamics, salt corrections, and modification adjustments.

General Questions

Q:What is OligoPool.com?

OligoPool.com is a free online platform providing scientifically accurate oligonucleotide design and analysis tools. All calculations run locally in your browser—your sequence data never leaves your device. We offer tools for Tm calculation, GC content analysis, secondary structure prediction, batch QC, and more.

Q:Is it really free?

Yes, completely free. No registration required, no paywalls, no premium features. All tools are freely available forever.

Q:Do I need to create an account?

No account required for basic use. However, creating a free account allows you to save calculation history, build sequence libraries, and access your saved data across devices.

Q:Is my sequence data secure?

Yes. All calculations run entirely in your web browser using JavaScript. Your sequences are never transmitted to our servers. You can verify this using browser developer tools.

Calculation Methods

Q:What method is used for Tm calculation?

We use the nearest-neighbor thermodynamics method based on SantaLucia (1998) unified parameters. This method calculates Tm by summing enthalpy (ΔH°) and entropy (ΔS°) contributions from all adjacent base-pair doublets in the duplex, then applying salt corrections for Na⁺, Mg²⁺, and dNTPs. Formula: Tm = ΔH° / (ΔS° + R ln(C/4)) - 273.15 + 16.6 log₁₀[Na⁺], where C is oligo concentration and R is the gas constant. This is the gold standard for 15-70nt oligos (±2°C accuracy) and is used by IDT, Thermo Fisher, and academic tools. For comparison with other methods or to calculate your own primers, use our Tm Calculator with customizable salt parameters.

Q:Why do different calculators give different Tm values?

Tm differences (typically ±2°C) are normal and can result from: 1) Different nearest-neighbor parameter sets (SantaLucia 1998 vs Breslauer 1986), 2) Different salt correction formulas, 3) Handling of terminal mismatches, 4) Assumptions about oligo concentration. Our calculator uses SantaLucia 1998 parameters with salt correction.

Method	Oligo Length	Accuracy	Best For
Wallace Rule (GC-based)	≤14 nt	±5°C	Quick estimates only
Nearest-Neighbor (SantaLucia 1998)	15-70 nt	±2°C	PCR primers, probes
Nearest-Neighbor (Breslauer 1986)	15-70 nt	±3°C	Legacy calculators
Salt-Adjusted NN	>50 nt	±2°C	Long oligos, gene synthesis

Table: Comparison of Tm calculation methods. SantaLucia 1998 is the current gold standard for most applications.

Q:How accurate are the calculations?

Tm calculations: ±2°C accuracy for unmodified DNA oligos 15-70nt under standard conditions (nearest-neighbor method). Accuracy degrades for: modified bases (LNA, PNA, 2'-O-Me), extreme GC content (<30% or >70%), very short oligos (<15nt), or mismatched salt concentrations. GC content: 100% accurate (simple base counting). Molecular weight: ±0.1% accuracy using exact molecular weights for incorporated nucleotides in DNA chains: A=313.2, T=304.2, G=329.2, C=289.2 g/mol (verified by Thermo Fisher, CUSABIO). These values account for phosphodiester bond formation. Secondary structure: ΔG predictions correlate well with experimental data but are model-dependent. Always validate critical designs experimentally. Use our Oligo Properties Calculator for comprehensive analysis.

Q:What salt concentrations should I use?

Salt concentrations significantly affect Tm calculations. Use these standard values based on your application:

Application	Na⁺ (mM)	Mg²⁺ (mM)	dNTP (mM)
Standard PCR	50	1.5-2.5	0.2-0.8
High-fidelity PCR	50	2.0	0.2
qPCR/Real-time PCR	50	3.0-5.0	0.2
Hybridization	50-100	0-5	0
Sequencing reactions	40-50	1.5	0.5

Note: Always check your specific enzyme/kit manufacturer's protocol for exact concentrations. These values represent typical industry standards.

Q:How do I calculate oligo resuspension concentration?

To resuspend lyophilized oligos to a target concentration: 1) Calculate molecular weight (MW): Use our Molecular Weight Calculator or synthesis report, 2) Determine nmoles: Check synthesis scale (typically 25-250 nmol from vendors), 3) Calculate volume: Volume (µL) = nmoles / (target concentration in µM) × 1000. Example: 100 nmol oligo to 100 µM = 100 / 100 × 1000 = 1000 µL (1 mL) TE buffer. For accurate quantification after resuspension, measure A260 (1 OD₂₆₀ unit ≈ 33 µg/mL for ssDNA), then calculate concentration using extinction coefficient (ε₂₆₀). IDT and other vendors provide these values on spec sheets.

Tool Usage

Q:Can I process multiple sequences at once?

Yes! The Batch Sequence QC tool supports up to 10,000 sequences. GC Content Analyzer and Format Converter also support batch processing. Upload FASTA or CSV files for bulk analysis.

Q:What sequence formats are supported?

We support FASTA, CSV, TSV, and plain text formats. The Format Converter tool can convert between formats with automatic detection and validation.

Q:How do I interpret secondary structure results?

Hairpins: ΔG < 0 kcal/mol indicates stable structure. Self-dimers: Check ΔG value—lower values indicate stronger dimers. Avoid sequences with ΔG < -5 kcal/mol for primers. Hetero-dimers: Check if your primer pairs form dimers. Use our Secondary Structure Predictor tool for detailed analysis, or see the User Guide for interpretation guidelines.

Q:What GC content is optimal?

For PCR primers: 40-60% is ideal. For probes: 50-60% works well. Avoid extremes: <30% GC may be unstable, >70% GC may cause secondary structures. Use our GC Content Analyzer to check your sequences, or follow the PCR Primer Design workflow for best practices.

Q:What is the best workflow for PCR primer design?

Follow this systematic workflow for reliable PCR primer design:

Parameter	Optimal Range	Why It Matters
Primer Length	18-25 nt	Sufficient specificity without excess cost
Tm (Melting Temp)	55-65°C	Standard PCR enzyme activity range
ΔTm Between Pairs	<5°C	Ensures both primers anneal efficiently
GC Content	40-60%	Balanced stability and specificity
GC Clamp (3' end)	1-2 G/C bases	Promotes stable 3' binding for extension
Runs (Repeats)	<4 identical bases	Prevents mispriming and slippage
3' End Stability	ΔG > -9 kcal/mol	Avoids strong self-complementarity
Hairpin ΔG	> -3 kcal/mol	Prevents self-folding that blocks annealing
Dimer ΔG	> -5 kcal/mol	Minimizes primer-dimer artifacts

Design Workflow:

Design initial primers following the parameter guidelines above
Calculate Tm: Use Tm Calculator with your PCR buffer conditions
Check secondary structure: Use Secondary Structure Predictor to identify problematic hairpins and dimers
Verify GC content: Use GC Analyzer for distribution analysis
Check specificity: BLAST primers against your target genome/database
Order and optimize: Start with gradient PCR (±5°C around calculated Tm)

For detailed guidance, see our PCR Primer Design Guide. These guidelines are based on industry standards and published best practices (Dieffenbach & Dveksler, 2003; Rychlik, 1995).

Troubleshooting

Q:The tool is not responding or very slow.

If a tool becomes unresponsive: 1) Wait 30 seconds (large batches may appear frozen). 2) Refresh the page and try with fewer sequences. 3) Clear browser cache (Ctrl+Shift+Delete). 4) Try a different browser (Chrome/Firefox recommended). 5) Report persistent issues to support@oligopool.com.

Q:I think the calculation result is wrong.

Double-check: 1) Input sequence (copy-paste errors?). 2) Parameters (salt concentration, temperature). 3) Compare with another calculator or published data. 4) For Tm, differences of ±2°C between calculators are normal due to method variations. Report calculation errors with example data to support@oligopool.com.

Q:The page won't load or displays errors.

Try: 1) Clear browser cache and cookies. 2) Disable browser extensions temporarily. 3) Try incognito/private browsing mode. 4) Update your browser to the latest version. 5) Check if JavaScript is enabled.

Q:I can't save my calculation history.

Ensure you're logged in. Check browser local storage is enabled. If problems persist, try logging out and back in. Contact support@oligopool.com if issues continue.

Data & Privacy

Q:Do you store my sequences?

No. Calculations run entirely in your browser. Sequences are only saved if you explicitly click"Save to Library" while logged in. Even then, saved sequences are encrypted in our database.

Q:Can I export my calculation results?

Yes. Most tools support CSV/Excel export. Batch QC results can be exported as comprehensive reports. Format Converter supports multiple export formats.

Q:How do I delete my account and data?

Go to Dashboard → Settings → Privacy → Delete Account. This permanently removes all your data including saved sequences and calculation history.

Q:Do you use cookies?

We use minimal cookies for authentication (if logged in) and basic website analytics (Google Analytics). No cookies are used to track your research data. See our Privacy Policy for details.

Advanced Usage

Q:Can I use these tools for commercial purposes?

Yes, all tools are free for commercial use. However, always validate critical designs independently. See our Terms of Service for details.

Q:How do I cite these tools in publications?

Cite the underlying scientific methods (SantaLucia 1998, etc.) rather than this website. See our Scientific References page for proper citations. If you want to acknowledge the website, you can mention"OligoPool.com" as a tool used.

Q:Are there API access or bulk download options?

Not currently. As an individual developer project, API access is not available. However, batch processing tools support large file uploads (up to 10,000 sequences).

Q:Can I import my calculation history from another account?

Not directly through the UI. However, if you have an exported CSV/JSON file, you can re-run those calculations while logged in to save them to your current account. For bulk imports or account merges, contact support at support@oligopool.com with your use case.

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