Notes
- While not “best-practice”, this protocol works well if you don’t need well-purified plasmids or if you don’t have a commercial plasmid mini-prep kit on hand.
- I would not recommend this for cloning work, however, it can be used for quick digests and for cloning vectors into E. coli or Agrobacterium.
Source
- Birnboim, H.C. and J. Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res, 1979. 7(6): p. 1513-23.
- Adapted from ACPFG protocol.
Materials
- Overnight culture containing plasmid for extraction.
- Benchtop centrifuge.
- 45°C Heat block (optional).
- 1.5 – 2 mL Eppendorf tubes.
- 70% Ethanol.
- 100% Isopropanol.
- Solutions:
- Solution I, II & III.
- RNAase H2O (50 µg / mL).
| Solution I | 500 mL | M |
|---|---|---|
| Glucose | 4.95 g | 50 mM |
| Tris-HCl, pH 8.0 | 1.15 g | 25 mM |
| EDTA | 3.33 mL of 0.5 M EDTA stock solution | 10 mM |
| ddH2O | to 500 mL | – |
| Solution II | 500 mL | M |
|---|---|---|
| NaOH | 4 g | 0.2 M |
| 1 % SDS | 25 mL of 20 % stock solution | – |
| ddH2O | to 500 mL | – |
| Solution III | 500 mL | M |
|---|---|---|
| Potassium acetate | 147 g) | 3 M |
| ddH2O | 400 mL | – |
| 100% acetic acid | bring to pH 4.8 (57.5 mL) | – |
| ddH2O | to 500 mL | – |
H2O containing RNase
- RNase concentration: 50 µg / mL in autoclaved ddH2O
- before preparation, RNase needs to be heated up 15-30 min at 95 º C to remove contaminating DNase. Store RNase-H2O at 4ºC. RNase from Fermantas and is already DNase free and does not need to be heated up.
Method
- Overnight culture of E. coli.
(E. coli grown overnight in 2.5 mL LB plus antibiotics in 37°C shaking incubator.) - Transfer 1.5-2 mL in Eppendorf tube.
- Centrifuge cells (benchtop centrifuge, max speed, 1 min).
- Discard supernatant.
- Re-suspend in 100 µL solution I (vortex or shaking incubator).
- Add 200 µL solution II.
- Add 150 µL solution III, invert tube carefully 6-8 times.
- Centrifuge for 10 – 15 min (benchtop centrifuge, max speed).
- Transfer supernatant in a fresh tube (do not take any of the white precipitate).
Following steps use the supernatant. - Add 750 µL Isopropanol, invert tubes. Incubate at room temperature for 15 min.
- Optional: Incubate at -20°C overnight to increase yields (and can go home early)
- Optional, optional: Incubate for 1 minute if in a rush. Yield will be lower.
- Centrifuge 10-15 min (benchtop centrifuge, max speed).
- Remove supernatant, keep white pellet (possible by inverting tube).
- Add 0.5 -1 mL Ethanol (70%) and vortex short (1 sec).
- Centrifuge 2-10 min (benchtop centrifuge, max speed).
- Remove supernatant completely with a pipette.
- Air-dry pellet or on heat block at 45°C for a few minutes.
- Re-suspend in 30 µL H2O containing RNase and incubate at 37°C for about 10 min.
- Freeze DNA at -20°C for storage.
