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Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Molecular Biology > DNA > DNA extraction
Author: Fanglian He, 2/5/2011, 28145 views, 23 Q&A

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits.

Materials and Reagents

  1. Luria-Bertani broth (LB) medium: Bacto-tryptone (BD), yeast extract (BD)
  2. RNAase (Invitrogen)
  3. Isopropanol (EM Science)
  4. Ethanol (VWR Chemical)

Equipment

  1. Table-top centrifuge

Procedure

  1. Grow bacterial (E. coli) culture in LB medium with appropriate antibiotics at 37 °C overnight (O/N) with shaking. For > 10 copies plasmid, 3 ml cell culture is usually enough.
  2. Transfer O/N culture to a 1.5 ml Eppendorf tube, and spin down cell culture (twice) at high speed for 1 min at table-top centrifuge.
  3. Discard the supernatant. To remove the liquid completely by upside down tube onto a piece of paper towel for a few seconds.
  4. Add 100 μl of resuspension solution (P1 buffer) into each tube, and vortex to completely resuspend cell pellet
  5. Add 100 μl of lysis solution (P2 buffer) and mix by gently inverting the tube 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial lysis has taken place.
  6. Add 150 μl of neutralizing solution (P3 buffer) and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
  7. Centrifuge the tubes at high speed for 10 min.
  8. Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5 ml Eppendorf tube with a 1 ml pipette.
  9. Add 2.5-3 volume of 200-proof cold ethanol (stores at -20 °C) to each tube and mix by inverting the tubes for a few times.
    Note: In order to increase DNA yield, you can keep the tubes at -20 °C for 30 min or longer before centrifuge (next step).
  10. Spin down plasmid DNA precipitate (transparency pellet) at high speed at 4 °C for 10 min.
  11. Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside-down on a piece of paper towel, then keep the tubes in a tube holder and air dry for 10-20 min. To dry faster, keep tubes at 37 °C heat blocker. DNA precipitate turns to white when dry.
  12. Resuspend the DNA pellet with 50 μl TE. To completely dissolve the pellet by pipetting solution several times.
    Note: lots of RNA is present in the DNA sample. So, for subsequent reaction, for example, to digest plasmid DNA, add 1-5 μl (1 mg/ml) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to resuspension solution with a final concentration of 1 mg/ml.

Recipes

  1. LB medium: 1% Tryptone, 0.5% yeast extract, 200 mM NaCl.
  2. Resuspension solution (P1 buffer): 50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0. Store at 4 °C.
  3. Lysis solution (P2 buffer): 0.2 N NaOH, 1% SDS. Store at room temperature.
  4. Neutralizing solution (P3 buffer): 3 M KOAc, pH 6.0. For 100 ml solution, 60 ml 5 M potassium acetate (49.07 g potassium acetate in 100 ml H2O), 11.5 ml glacial acetate and 28.5 ml H2O. Store at room temperature.
  5. TE: 1 mM EDTA, 10 mM Tris-HCl, pH 8.0.
    Note: P1 P2 P3 buffers from QIAGEN also works well.

References

  1. Birnboim H.C., Doly J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6): 1513-23.
  2. Birnboim H.C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods in Enzymology 100: 243-55.


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