Click on individual wells to toggle active PCR targets, or use the quick templates. The master mix calculations will automatically scale.
Analysis Results
How Colony PCR Works
Colony PCR is a rapid, high-throughput technique used to screen bacterial colonies (typically E. coli after transformation) for the presence of a target plasmid or gene insert. Instead of performing a tedious plasmid purification (mini-prep) on dozens of cultures, you can lyse bacterial cells directly in the PCR reaction.
Standard vs High-Throughput Colony PCR
Standard Colony PCR: Ideal for smaller batches of screens (8-24 colonies). Usually done in strip tubes. Primers are selected to either amplify the insert directly (insert-specific) or span across the cloning site (backbone-specific) to easily differentiate empty plasmids from positive clones based on fragment length.
High-throughput 96-well: Essential when screening large numbers of candidates. Toggling active wells on our interactive layout automatically updates your scaling factor, ensuring you make exactly the correct master mix volume, accounted for multi-channel pipetting overage (typically 10-15%).
Cell Lysis Protocol Guide
1. Aliquot your prepared PCR Master Mix into tubes or plate.
2. Pick a single colony gently with a sterile tip, touching it lightly.
3. Swirl tip directly in the reaction volume to transfer cells.
4. Alternative (Highly Recommended): Resuspend colony in 10µL H₂O. Heat at 95°C for 5 min. Spin down, then use 2µL of supernatant. This prevents PCR inhibition by bacterial cellular debris!
5. Program a long initial denaturation (95°C for 5-10 min) to lyse bacteria and expose DNA.
Crucial Colony PCR Tips
- Don't pick too much: A tiny, barely visible smudge of bacteria is more than enough. Picking too much biomass will inhibit the polymerase and lead to failed reactions.
- Keep a backup: Touch the tip onto a fresh agar grid plate (backup master plate) before swirling it in the PCR mix so you can recover positive colonies the next day!
- Control reactions: Always run a negative control (water template or host bacterium without plasmid) and a positive control (purified plasmid if available).