Recovering Plasmid DNA from Bacterial Cultures without Using Kits

Introduction

Many molecular biology techniques, such as complete plasmid DNA sequencing, require highly purified and concentrated plasmid DNA, and bacterial plasmids are widely used as cloning vectors for DNA recombination. After constructing a new plasmid, its size and restriction endonuclease spectrum can be determined by gel electrophoresis. This article will introduce two effective protocols for plasmid extraction without using kits.

Protocol A

Equipment

• Desktop microcentrifuge
• Desktop vortexer

Reagents

• Denaturing solution
• Renaturing solution
• 100% ethanol or isopropanol
• 70% ethanol
• TE buffer or water

Protocol

1. Grow an overnight culture of bacteria.

2. Centrifuge the culture to precipitate bacteria prior to DNA preparation.

3. Remove the supernatant and resuspend the bacteria in buffer.

4. Add a denaturing solution to the resuspended bacteria in order to cause bacteria lysis and release their contents.

5. Add a renaturing solution to the denatured bacteria in order to precipitate protein and genomic DNA and leave plasmids free in solution.

6. Precipitate the protein and genomic DNA by centrifugation, and remove the supernatant that contains plasmids.

7. Add either ethanol or isopropanol to precipitate the plasmid DNA.

8. Precipitate the DNA using centrifuge or apply the solution to a column that will bind to the DNA.

9. Wash the pellet or column with 70% ethanol to remove excess salt.

10. Resuspend the DNA pellet, or elute the DNA off of the column using water or a neutral buffer such as TE.

Protocol B: the alkaline extraction method

Equipment

• Eppendorf-type (1.5 ml) polypropylene tubes
• A bench-top centrifuge capable of generating 8-10,000 xg

Reagents

• Solution I: Lysozyme solution- 2 mg/ml lysozyme, 50 mM glucose, 10 mM CDTA, 25 mM Tris-HC1 (pH 8.0)
• Solution II: Alkaline SDS solution- 0.2 N NaOH, 1% sodium dodecyl sulfate (SDS)
• Solution II: High salt solution-3 M sodium acetate (pH 4.8)
• 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8)
• Cold ethanol

Protocol

1. Grow an overnight culture of bacteria.

2. Transfer 0.5 ml culture to 1.5 ml Eppendorf tube for plasmid extraction.

3. Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.

4. Remove the supernatant, add 100 ul of Solution I, resuspend the cell pellet, and incubate at 0 °C for 30 min.

5. Add 200 ul of Solution II and gently rotate. The suspension should become almost clear and slightly viscous. Keep the tube at 0 °C for 5 min.

6. Add 150 ul of Solution III and gently mix the contents of the tubing by inversion for a few seconds, during which time a DNA clot formed. The tube is kept at 0 ° C for 60 min.

7. Centrifuge for 5 min, at 10,000 xg.

8. Remove supernatant and transfer it to a second centrifuge tube, add 1 ml of cold ethanol and held the tube at -20°C for 30 min.

9. Collect the precipitate by centrifugation for 2 min.

10. Remove the supernatant, and dissolve the pellet in 100 ul of 0.1 M sodium acetate/0.05 M Tris-HCl (pH 8) and reprecipitate it with 2 volumes of cold ethanol, for 10 min at -20℃.

11. Centrifuge for 5 min, at 10,000 xg, and dissolve pellets in 40 μl of water.

At CD Genomics, we provide complete plasmid DNA sequencing to help you investigate the evolution of plasmids, their modular structure and the existence of hot spots for the insertion of accessory genes.

Additional Readings

The Methods for DNA Extraction and Purification

High-throughput Sequencing Sample Submission Guidelines

Reference:

1. 1. Bimboim H C, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 1979, 7(6):1513-1523.