Alkaline lysis has now been used for almost 30 years to isolate plasmid DNA from E. coli (Birnboim and Doly, 1979). In the process, exposure of bacterial suspensions to the strong detergent sodium dodecyl sulfate (SDS) under alkaline conditions opens the cell wall, denatures chromosomal DNA and proteins, and releases plasmid DNA into the supernatant. Although the alkaline solution completely disrupts base pairing, the strands of closed circular plasmid DNA can again form a double-strand when the pH is returned to neutral and the exposure time to denaturing conditions is not too long. During lysis, bacterial proteins and denatured chromosomal DNA become trapped in large complexes that are coated with SDS. Replacing the sodium by potassium e ciently precipitates these complexes from solution since potassium dodecyl sulfate is much less soluble than SDS. After the precipitate has been removed by centrifugation, the plasmid DNA can be recovered from the supernatant.
Further puri cation was achieved using commercially available chromatography resins in gravity fast ow columns (Qiagen, Genomed). These columns contain a porous silica- based resin coated with positively charged diethylaminoethanol-(DEAE)-groups. Double- stranded DNA readily binds to the silica resins due to dehydration of the phosphodiester backbone, which allows the exposed negatively charged phosphate residues to bind to the
DEAE-groups. Once adsorbed, RNAs, proteins, and carbohydrates are removed from the columns by washes with low salt buers, while DNA remains immobilized. Finally, the puri ed plasmid DNA is eluted from the column with high salt buer and concentrated by alcohol precipitation.
Minipreparation Plasmid DNA was isolated from small-scale bacterial cultures (2 mL) to rapidly screen large numbers of colonies for plasmids containing the correctly inserted DNA fragments. Screening was performed by digesting isolated plasmid DNA with suit- able restriction endonucleases to detect the presence of the mutations introduced.
2 mL of 2xYT medium containing 200 µg/mL ampicillin and 20 µg/mL chlorampheni- col was inoculated with a single colony of transformed bacteria. Cultures were incubated overnight at 37◦C with vigorous shaking. To harvest cells, 1.5 mL of a culture were trans-
ferred to a reaction tube and centrifuged at maximum speed for 2 min in a microfuge. The unused portion of the original culture was stored at 4◦C for later inoculation of in-
termediate scale plasmid preparations. After centrifugation, the medium was completely removed and the bacterial pellet was resuspended in 200 µL ice-cold buer P1 and vor- texed until completely dispersed. Each suspension was supplemented with 400 µL freshly prepared buer P2 and the contents mixed by inverting rapidly 5-10 times. Denaturation was allowed to continue for 5 min at room temperature before neutralizing the lysates with 300 µL of ice-cold buer P3. Tube contents were mixed by inverting until no remainder of the viscous lysate was left. After incubation on ice for 5 min, tubes were centrifuged at maximum speed for 5 min at 4◦C and the supernatant transferred to a fresh tube. To
recover the plasmid DNA, nucleic acids were precipitated from the supernatant by adding 600 µL of isopropanol (see 4.3.5).
Buer P1 Buer P2 Buer P3
25 mM Tris-HCl pH 8.0 200 mM NaOH 3 M potassium acetate
50 mM glucose 1 % (w/v) SDS 2 M acetic acid
10 mM EDTA
Midipreparation Intermediate-scale plasmid DNA preparations were performed using the Plasmid Midi Kit (Qiagen, Hilden) or the JetStar 2.0 Plasmid Kit (Genomed, Löhne). With these kits, crude lysates of bacteria prepared by alkaline lysis are further puri ed by anion exchange chromatography. The plasmid DNA yield is su ciently pure for enzymatic manipulation as well as eukaryotic cell transfection.
50 mL of LB medium, supplemented with ampicillin and chloramphenicol (see 4.3.10), were inoculated with 20 µL of the unused portion of original minipreparation cultures, which had been screened and identi ed to contain the desired recombinant plasmid. Al-
ternatively, a chosen glycerol stock was streaked out on LB agar plates containing the appropriate antibiotic. Plates were incubated overnight and a single colony of bacteria was inoculated into 50 mL LB medium for subsequent midipreparation. Inoculated cul- tures were incubated overnight at 37◦C with vigorous shaking. Cultures were transferred
to 50 mL tubes and the bacteria harvested at 5000 rpm for 20 min at 4◦C. The medium
was completely removed. The pellets were completely dispersed in 4 mL of ice-cold buer E1, 4 mL of lysis buer E2 were added, and the tube contents mixed by inverting 4-5 times. Lysis was allowed to continue for 5 min before transferring the samples to 15 mL tubes that already contained 4 mL of neutralization buer E3. Tubes were inverted several times until no remainder of the viscous lysate was left. The resulting white precipitate was removed by centrifugation at 16000 rpm for 10 min at room temperature and the clear supernatant applied to equilibrated fast ow columns provided with the kits. The lysate was allowed to pass through the column by gravity ow. Columns were washed two times with 10 mL washing buer, before the DNA was recovered with 5 mL elution buer. 3.5 mL isopropanol were added to precipitate the puri ed plasmids, and the precipitate collected by centrifugation (see 4.3.5). Due to the slow, autocatalytic acidic hydrolyza- tion of its backbone phosphodiester bonds in aqueous solution, isolated DNA was kept at -20◦C in TE buer for long-term storage.
Buer E1 Buer E2 Buer E3
50 mM Tris-HCl pH 8.0 200 mM NaOH 3.1 M potassium acetate
10 mM EDTA 1 % (w/v) SDS pH 5.5 with acetic acid
100 µg·mL-1 RNase A
Buer E4 Buer E5 Buer E6
600 mM NaCl 800 mM NaOH 1250 mM NaOH
100 mM sodium acetate 100 mM sodium acetate 100 mM Tris-HCl pH 8.5 0.15 % Triton X-100 pH 5.0 with acetic acid
pH 5.0 with acetic acid