Isolation of Plasmids from E.

coli by Boiling Lysis

Sabine Ehrt and Dirk Schnappinger Introduction The boiling lysis procedure (1) is quick to perform and, therefore, especially suitable for screening large numbers of small-volume Escherichia coli cultures. It is described with different adaptations in a variety of protocol books (2,3). The quality of the isolated plasmid DNA is lower than that from an alkaline lysis miniprep, but it is sufficient for restriction analysis. The bacteria are lysed by treatment with lysozyme, Triton, and heat. The chromosomal DNA remains attached to the bacterial membrane and is removed by centrifuga-tion. The plasmid DNA remains in the supernatant from which it is then precipitated with isopropanol. The boiling lysis procedure is not recommended when isolating plasmids from E. coli endA+ strains that express endonuclease A, such as HB101 and the JM100 series. Contamination of plasmid preps by endonuclease A, which is not completely inactivated by boiling, results in plasmid degradation during subsequent incubation in the presence of Mg2+, (e.g., during digestion with restriction enzymes). This problem can be overcome by including an extraction with phenol : chloroform to remove contaminating endonuclease from the plasmid prep. Alternatively, the alkaline lysis method can be used (see Part 8). Materials Growth of E. coli 1. Luria-Bertani (LB) medium: 5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone. Autoclaved. 2. Appropriate antibiotics. Plasmid Isolation 1. STE solution: 8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0). Autoclave and store at 4C.

2. STET solution: 8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 5% (w/v) Triton X100. Filter-sterilize and store at 4C. 3. 10 mg/mL Lysozyme in 10 mM Tris-HCl (pH 8.0) (see Note 1). 4. Phenol : chloroform (1 : 1). 5. 100% Isopropanol. 6. 70% Ethanol. 7. TE buffer: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0). Autoclave and store at room temperature. 8. 10 mg/mL RNase A, DNase-free. Methods Growth of E. coli 1. Inoculate 3 mL of sterile LB medium containing the appropriate antibiotic with a single bacterial colony. 2. Grow with shaking at 37C overnight. Plasmid Isolation 1. Centrifuge 1.5 mL of culture for 20 s in a microcentrifuge at maximum speed to pellet the bacteria. Remove the supernatant as completely as possible (see Note 2). 2. Optional wash step (see Note 3): Resuspend the cell pellet in 0.5 mL STE solution. Centrifuge again and remove the supernatant. 3. Resuspend the bacterial pellet in 350 4. Add 25 of STET by vortexing.

of lysozyme solution and mix by vortexing for 3 s.

5. Place in a boiling water bath for 1 min.

6. Centrifuge for 10 min in a microcentrifuge at maximum speed. 7. Remove the viscous pellet with a sterile toothpick (see Note 4). 8. Optional extraction with phenol: chloroform (see Note 5): Add an equal volume of phenol: chloroform (1:1) and mix by vortexing for 5 s. Centrifuge for 1 min in a micro-centrifuge at maximum speed to achieve phase separation. Transfer the top aqueous phase to a clean tube. 9. Mix the supernatant with 450 of isopropanol by vortexing and incubate at room temperature for 5 min. 10. Centrifuge for 10 min at 4C in a microcentrifuge at maximum speed. 11. Carefully remove the supernatant. Add 1 mL of 70 % ethanol and centrifuge for 2 min at 4C in a microcentrifuge at maximum speed. Remove the supernatant as completely as possible and let the pellet air-dry for 10 min (see Note 2). 12. Dissolve the pellet in 50 Plasmid Analysis Gel Analysis Analyze the DNA by agarose gel electrophoresis as described in Part 20. It is recommended that undigested supercoiled plasmid DNA be analyzed to verify the integrity of the DNA and to assess the content of chromosomal DNA (see Notes 7 and 8). In addition, analyze the DNA by cleavage with restriction enzymes. Use 1 of the plas-mid preparation in the case of high-copy-number plasmids (such as pUC derivatives) and 3 or more in the case of medium- or low-copy-number plasmids. If the DNA is resistant to cleavage with restriction enzymes, extract the isolated plasmid DNA with phenol : chloroform and precipitate again with ethanol (see Subheading 3.2.). The concentration of the plasmid DNA can be roughly estimated by comparing it to a plasmid of known concentration. Run 0.1 of the plasmid standard and several amounts (e of TE buffer containing 20 ng/mL RNase A (see Note 6).

<< BACK NEXT >>

NAVIGATION