Plasmid DNA Extraction
Related: DNA Extraction · Plasmid · Plasmid and Mobile Element Analysis · Whole-Genome Sequencing
1. Principle
Plasmid extraction (miniprep/midiprep/alkaline lysis) selectively recovers extrachromosomal circular DNA by denaturing genomic DNA and precipitating or binding plasmid DNA for cloning, PCR, or sequencing of mobile elements.
2. Step-by-Step Procedure (conceptual)
- Grow isolate / transformant under selection if applicable.
- Alkaline lysis — chromosomal DNA denatures; supercoiled plasmids renature.
- Neutralize; precipitate proteins/chromosomal debris.
- Bind plasmid DNA (silica/beads) → wash → elute.
- Optional: linearization QC on gel; sequence or transform.
3. Interpretation / QC
- Yield depends on copy number (high-copy vs low-copy Inc types).
- Large low-copy MDR plasmids may need gentle handling or long-read WGS from total DNA instead.
- Pitfalls: Chromosomal contamination; nicking; shearing of mega-plasmids.
4. Clinical / Reference Use Cases
- Research characterization of resistance plasmids.
- Reference labs investigating plasmid outbreaks (often total-DNA long-read is preferred clinically).
- Cloning diagnostic controls.
5. Comparison with Other Methods
| Method | Recovers |
|---|---|
| Plasmid prep | Enriched plasmid DNA |
| Total DNA Extraction | Chromosome + plasmids |
| Long-Read and Hybrid Bacterial Assembly | Complete plasmid maps from total DNA |
6. Mnemonic / Visual Aid
Alkaline shock keeps circles, dumps the chromosome.
Active Recall
- Why do mega-plasmids disappear from harsh minipreps?
- When prefer long-read WGS over classical plasmid prep?
- What does copy number mean for yield?