Last updated: April 21, 2026

How to Design an Oligo Pool Before Ordering

Use this page when you are turning a CRISPR, NGS, mutagenesis, or gene-assembly idea into an order-ready pool. It walks through application choice, sequence rules, batch QC, vendor selection, and submission prep so you can catch avoidable failures before ordering.

Scientist collaborating over DNA sequence alignments and design software

Planning and validating library sequences before array synthesis.

What Is an Oligo Pool?

An oligo pool (oligonucleotide pool) is a collection of thousands to millions of unique DNA sequences synthesized simultaneously on a microarray chip, then cleaved and delivered as a mixed solution.

๐Ÿงช

Individual Oligo

$5-50/oligo
1 sequence โ€ข 1-3 days
๐Ÿงฌ

Oligo Pool

Much lower/oligo
2K-1M+ sequences โ€ข 2-6 biz days
๐Ÿ’ฐ

Savings

Orders of magnitude
per sequence โ€ข at scale

5-Step Pre-Order Design Checklist

1

Choose Your Application

Different applications have different pool requirements. A CRISPR sgRNA pool usually needs 10K-200K short oligos (80-110 bp). Gene assembly needs fewer but longer oligos (150-350 bp). NGS panels typically need 120 bp capture probes.

Design a CRISPR sgRNA Oligo Pool โ†’Design a Custom NGS Panel Oligo Pool โ†’Design a Mutagenesis Oligo Pool โ†’Assemble Synthetic Genes from an Oligo Pool โ†’
2

Design Your Sequences

Follow the sequence design rules: keep GC content between 30-70%, avoid homopolymers (>4 bases), check for secondary structures (ฮ”G > -3 kcal/mol), and maintain length uniformity within ยฑ10% across the pool.

GC Analyzer โ†’Structure Predictor โ†’
3

Screen for Synthesis Problems

Run all sequences through batch QC to identify problematic oligos before ordering. This step catches 5-15% of sequences that would fail synthesis or under-represent in the pool.

Batch QC โ†’
4

Select a Vendor

Compare vendors by pool size capacity, max oligo length, cost per oligo, NGS QC inclusion, and turnaround time. Each vendor has different strengths for different applications.

Vendor Comparison โ†’
5

Submit & Validate

Format your sequences as FASTA or CSV, submit order, and after synthesis, validate pool quality by NGS. Check representation (โ‰ฅ90%), dropout (<10%), and uniformity (CV <3-fold).

Format Adapter โ†’Uniformity Estimator โ†’

Design Rules Quick Reference

RuleAcceptableAvoidTool to Check
GC Content30-70%<25% or >75%Check โ†’
Homopolymersโ‰ค4 basesโ‰ฅ5 consecutive same baseCheck โ†’
Secondary Structureฮ”G > -3 kcal/molStrong hairpins (ฮ”G < -5)Check โ†’
Length Uniformityยฑ5-10% of target length>20 bp variationCheck โ†’
Repeatsโ‰ค4 bp repeat unitsLong tandem repeatsCheck โ†’

For detailed design constraints, see our complete design rules reference.

Common Pre-Order Mistakes

โœ•

Skipping pre-synthesis QC

Always run Batch QC before ordering. 5-15% of sequences have synthesis-problematic features.

โœ•

Ordering oligos too long for the platform

Check vendor max length (200-350 bp). Longer oligos have exponentially higher error rates.

โœ•

Not including controls

Add 500-1,000 non-targeting sequences for CRISPR, or wild-type sequences for mutagenesis.

โœ•

Ignoring secondary structures

Hairpins with ฮ”G < -5 kcal/mol cause synthesis dropout. Screen with Structure Predictor.

โœ•

Using wrong file format

Each vendor has specific format requirements. Use our Format Adapter to avoid rejection.

Frequently Asked Questions

What is an oligo pool?โ–พ
An oligo pool is a collection of thousands to millions of unique oligonucleotide sequences synthesized together in a single manufacturing process. Instead of ordering each oligo individually, array-based synthesis produces them all at once at a significantly lower per-oligo cost, delivered as a mixed pool.
What applications use oligo pools?โ–พ
CRISPR library screening (70K-200K sgRNAs), NGS target capture panels (2K-400K probes), gene assembly (10-300 overlapping fragments), deep mutational scanning (DMS), MPRA reporter assays, and DNA data storage. CRISPR screening is the largest application by volume.
What is the maximum oligo length in a pool?โ–พ
IDT oPools: up to 350 nt. Twist Bioscience: up to 300 nt (standard pools). Agilent SurePrint: up to 230 nt. GenScript GenTitan: up to 170 nt. Synthesis quality decreases with length โ€” keep oligos under 200 bp when possible for best results.
How do I submit my oligo pool order?โ–พ
Prepare a FASTA or CSV file with unique sequence IDs and sequences. Most vendors accept FASTA format. Check vendor-specific requirements (max length, character restrictions, minimum pool size). Use our Vendor Format Adapter to convert between formats.
How long does oligo pool synthesis take?โ–พ
Typical turnaround: 2-6 business days from order submission to shipment (varies by vendor). Twist: 2-4 business days. Agilent: 3-6 business days. GenScript: 3-5 business days. Rush options may be available โ€” contact vendor.

Next Pages to Open

Review Oligo Pool Design Rules

Check the technical constraints that prevent synthesis failures.

Compare Oligo Pool Vendors

See which vendor best fits your pool size, QC needs, and turnaround.

Estimate Oligo Pool Cost

Estimate pool cost by application, scale, and vendor fit.

Read Oligo Pool QC Metrics

Understand uniformity, dropout, error rate, and Gini before or after ordering.

Choose Between Array and Column Synthesis

Compare array-based and column-based synthesis trade-offs.

Troubleshoot Oligo Pool Problems

Review common pool problems and how to fix them.