Instruments
- Undergrad + liquid-handling module
- Thermocycler (annealing, incubation)
- Magnetic module for reaction clean-up
- Plate reader for colony/OD screening (optional)
- Onboard cameras enabled for vision verification
Input requirements
| Parameter | Target |
|---|---|
| Input | Annealed guide oligo duplex |
| Vector | Digested/linearized CRISPR backbone |
| Selection | Antibiotic per vector marker |
Labware
- PCR plate
- Reaction/vector plate
- Selection plates
- Reagent reservoirs
- Filter tips
Reagents
- Guide oligo pairs (top/bottom)
- Annealing buffer
- Digested CRISPR vector
- Ligase & buffer
- Competent cells
- Recovery media
- Selection antibiotic
- SPRI beads (optional clean-up)
Design and order oligos before the run. Keep competent cells cold until the transformation step and follow the strain's heat-shock or electroporation parameters. Ligation and transformation efficiencies are construct-dependent — validate a positive control alongside new guides.