•Small scale – minipreps – 1-5 ml culture –Yield – 5 to 100 mg plasmid, suitable for restriction analysis, sequencing, PCR
•Medium Scale – Midipreps – 10-50 ml culture –Yield – 100 to 1000 mg, suitable for all of the above plus transfection to mammalian cells, in vitro transcription, etc.
•Large scale – Maxipreps – 100-1000 ml culture –Yield – 0.5 to 5 mg, suitable for all of the above
Traditional Plasmid Mini Prep Protocol
Pellet cells from overnight culture
1. Resuspend pellet in 100 ml Solution I – Glucose, Tris (RESUSPENSION SOLUTION)
Purpose – keep cells alive during processing of samples
2. Add 200 ml Solution II – NaOH, SDS (CELL LYSIS BUFFER- make sure precipitate is dissolved)
Very high pH plus detergent – get limited cell lysis
3. Add 150 ml Solution III – Potassium Acetate (NEUTRALIZATION BUFFER)
High salt, acetate buffer neutralizes pH – Get flocculate precipitate
4. Remove precipitate by centrifugation
Genomic DNA, cellular proteins removed in the precipitate
5. Phenol-Chloroform Extraction/Isopropanol Precipitation
6. RNase Digestion
7. Phenol-Chloroform Extraction/Isopropanol Precipitation
