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E. coli Plasmid DNA MiniPrep Protocol

Introduction

The isolation of plasmid DNA from E. coli using an alkaline lysis is a well-established method. E. coli with plasmid is cultured in media with antibiotics to a high cell density, harvested, and then lysed with a SDS/NaOH solution. Rapid acidification using concentrated potassium acetate causes the precipitation of protein and chromosomal DNA. Plasmid DNA, which is supercoiled, remains in solution and can be captured on a silica spin column. The plasmid DNA is washed with an ethanol solution and then eluted in water or TE buffer.

Material

  • Microfuge tubes
  • Resuspension buffer (50 mM Tris HCl, pH 8, 10 mM EDTA, 100 µg/ml RNase A)
  • Lysis buffer (0.2 N NaOH, 1% SDS)
  • Neutralization buffer (3/5 M Potassium acetate, pH 6)
  • Spin columns (Product Number SSC 100-01)
  • Isopropanol
  • Wash buffer (70% Ethanol)
  • Elution buffer (water or TE buffer- 10 mM Tris, pH 8, 1 mM EDTA)

Equipment

  • Vortexer
  • Centrifuge

Protocol

  1. Culture E. coli with plasmid in LB media with antibiotic selective pressure, overnight on a shaker at 37°C.

  2. Pellet 1.5 ml of bacterial culture in a microfuge tube by centrifuging for 2 minutes at 10,000 rpm.

  3. Decant the supernatant and add 200 µl of the resuspension buffer. In order to resuspend the pellet you may have to vortex.

  4. Add 250 µl of the lysis buffer, invert the tube 10 times to mix thoroughly. The solution should become clear and viscous.

  5. Add 350 µl of the neutralization buffer, invert the tube 10 times or until a precipitate forms. The precipitate is a mixture of protein and chromosomal DNA.

  6. Centrifuge the tube for 10 minutes at 10,000 rpm. Transfer the supernatant to a microfuge tube and add 0.7 isopropanol. Incubate at -20°C for 15 minutes.

  7. Transfer the solution to a spin column.

  8. Centrifuge the spin column for 1 minute at 7,000 rpm. Discard the flow through.

  9. Add 400 µl of the wash buffer and centrifuge for 1 minute at 7,000 rpm. Discard the flow through. Repeat this step.

  10. Centrifuge for an additional 2 minutes at 10,000 rpm to remove residual wash buffer.

  11. Transfer the column to a clean microfuge tube. Add 50 µl of elution buffer and centrifuge for 1 minute at 10,000 rpm.

Results

Table 1: Eluted plasmid was read on a DeNovix spectrophotometer, to determine concentration. The machine was blanked with DI water.

Sample Name Concentration (ng/µl) 260/280 260/230
E. coli DH5α with pSVβ 1143.41 2.06 2.38

Figure 1: The isolated plasmid under went an enzymatic digest with HindIII (sourced from NEB) and was run on an Agarose Gel with Ethidium Bromide, along side two lanes of digested Lambda ladders (Click to enlarge the image).