Tutorial

What it does

Designs primers to amplify DNA fragments that can be assembled together using Gibson assembly.

Input

• List of DNA fragments (sequences)
• Optional: homology regions between fragments

Process

• Analyzes junctions between consecutive fragments
• Designs PCR primers that add homology overhangs to each fragment
• Optimizes overlap sequences for:
  • Length (15-40bp ideal)
  • Melting temperature (52-70°C)
  • GC content (40-60%)
  • Uniqueness (avoiding repeats)
  • Secondary structures (hairpins, dimers)

Output

• Visualization: Circular construct showing all fragments, overlaps, and primers
• CSV Tables:
  • Primer characteristics (sequence, Tm, GC%, secondary structures, quality scores)
  • Overlap characteristics (sequence, length, Tm, quality scores)
  • Primer order sheet (ready for IDT/Sigma)
  • PCR protocols for 3 polymerases (SuperFi II, Q5, KOD One)
• GenBank file: Annotated map with fragments, overlaps, and primers
• Expert explanation: LLM analysis of design quality, potential issues, and optimization tips

Use case

Building a new plasmid from synthesized gene fragments or PCR products.

What it does

Designs a Gibson assembly strategy to extract specific regions from existing plasmids in your inventory and combine them into a target construct.

Input

• Target construct description (what you want to build)
• Inventory of available plasmids with annotations
• Desired features/genes to extract

Process

• Searches inventory for plasmids containing desired features
• Plans extraction strategy (which regions to amplify from which plasmids)
• Designs primers to:
  • Amplify exact regions needed from source plasmids
  • Add homology overhangs for Gibson assembly
  • Create the final target construct
• Optimizes assembly for minimal number of PCR reactions

Output

• Same comprehensive outputs as "Gibson from fragments"
• Additional: Extraction plan showing which primers amplify which regions from which source plasmids

Use case

Building a new plasmid by mixing and matching parts from your existing plasmid collection (e.g., taking a promoter from plasmid A, gene from plasmid B, and resistance marker from plasmid C).

What it does

Designs optimal PCR primers to amplify specific regions from DNA templates.

Input

• One or more template sequences
• Optional: excluded region to amplify around (primers will flank this region)
• Product size constraints (min/max)
• Optional: Tm preferences

Process

• Uses Primer3 algorithm to design primer pairs
• For each template, finds primers that:
  • Flank the excluded region (if specified)
  • Produce desired amplicon size
  • Have matched melting temperatures (58-64°C ideal)
  • Minimize secondary structures
  • Avoid mispriming sites
• Calculates optimal PCR conditions for different polymerases

Output

• Visualization: Linear template(s) showing primers, excluded regions, and amplicons
• CSV Tables:
  • Primer characteristics (sequence, length, Tm, GC%, hairpin/dimer Tms, mispriming sites)
  • Amplicon characteristics (size, min Tm, penalty scores)
  • Gel electrophoresis setup (agarose %, voltage, run time, recommended ladder)
  • Primer order sheet
  • PCR protocols for 3 polymerases with calculated annealing temps and extension times
• GenBank files: One .gb file per template with annotated primers and amplicons
• Expert explanation: LLM analysis focused on primer quality, Tm matching, secondary structures, and polymerase selection

Use cases

• Amplifying a gene from genomic DNA or a plasmid
• Verifying insert presence (colony PCR)
• Adding restriction sites or tags to a sequence
• Batch primer design for multiple targets (e.g., screening multiple genes)