Plasmid Isolation- Traditional Plasmid Mini Prep Protocol

•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

Binod G C

I'm Binod G C (MSc), a PhD candidate in cell and molecular biology who works as a biology educator and enjoys scientific blogging. My proclivity for blogging is intended to make notes and study materials more accessible to students.

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