This method provides very pure DNA and contains only supercoiled plasmid DNA while nicked (relaxed circled) DNA is separated. A 250 ml bacterial culture was used. Harvest:
Notably, care was taken that the culture was not overgrown. Optimum was around 90% confluency. Spin down bacteria at 5000 x g for 20 minutes and drain off supernatant, re-suspend the pellet in 10 ml Qiagen buffer1 with RNAse. After complete re- suspension, 10 ml Qiagen buffer 2 for lysis was added, tube shaked gently and incubated for exactly 5 minutes. 10 ml of cold Qiagen buffer 3 (kept on ice) was addd, and mixed well. The mixture was filtered through a folded paper filter pre-wetted with clean water into a 50 ml tube (volume around 25 ml, some rest may stay in the filter). DNA was precipitated by adding 20 ml Isopropanol to the filtrate (1:1), mixed, and followed by a centrifugation step at 8000 x g for 1 hour at 4°C. Supernatant was drained off, the white pellet was washed carefully with 70% EtOH and spun again for 10 minutes. After draining off EtOH the wet pellet was dissolved in 15 ml TE pH 8.0. The pH of the solution should be around pH 8 (otherwise adjust pH).
Phenol extraction (may be omitted):
The Pour DNA solution was poured into a Phase Lock Heavy 50 ml tube, 7.5 ml of phenol equilibrated with TE and 7.5 ml Chloroform was added and shook well. Then the solution was spun at 1500 x g at RT for 20 min in a swing out rotor. The upper, aequeous phase was poured into a new 50 ml tube and 1.5 ml of 3 M Sodium Acetate pH 5.2 and 15 ml Isopropanol was added for DNA precipitation, mixed well and centrifuged at 8000 x g for 1 hour at 4°C. The supernatant was drained off and the pellet was washed with 70 % EtOH and spun again for 10 minutes. Rests of EtOH were removed, the DNA pellet was air-dried until transparency. Dry pellets may be stored at -20°C.
CsCl-gradient preparation and ultracentrifugation:
The dry pellet was dissolved completely in 8.0 ml TE. The DNA solution (8.0 ml) was added to 8.80 g CsCl in a 50 ml tube and the salt was dissolved completely (solution will get cold). 800µl of saturated EtBr was added and the solution was mixed until a fine precipitate was formed. Samples were warmed up to 37°C for 15 minutes in the water bath and centrifuged at full speed in a swing out rotor for 10 minutes so that the precipitate was clinged to the surface and the wall of the Falcontube. (Phenolextraction
6 Virus – vector construction and production 131
may be omitted but then more precipitate will be formed!). The supernatant was filled into an 11.2 ml OptiSeal tube (Beckmann) until the meniscus touched the basis of the tube’s neck. Tubes were balanced precisely to 0.0 g for the following ultracentrifugation step: 65.000 rpm for 5 hours and 30 minutes at 20°C at maximum acceleration and slow brake settings. Start of the centrifuge was delayed so that the spin ended at the next working day. After centrifugation finished visible DNA bands were immediately extracted.
Extraction of the EtBr from the rotor tube:
For harvest of the DNA band, a UV lamp in vertical direction and a dark room are required, as well as skin protection (i. e. wear lab coats, nitril gloves and face protection).
When the centrifuge stopped, tubes were removed from the rotor as soon as possible, and the tube was fixed in front of the UV-lamp in a holder. The tube’s shoulder was pierced with a canula (around 22 G) for ventilation of the tube when DNA-band was withdrawed. The tube was put in a fitting clamp, so that the band was visible halfway down the tube. Now the second needle (around 22 G) was put into the tube shortly under the DNA band. The UV-lamp was used to check the height of the DNA band (upper band: nicked DNA, lower band: super-coiled DNA). The needle was connected to a 2 ml syringe and as much DNA was sucked as possible by moving the syringe up and down. Content of the syringe was transferred into a 15 ml tube. DNA can be stored at that stage at room temperature overnight under light protection.
Removal of EtBr from the DNA:
An equal volume of n-Butanol saturated with TE was added to each of the DNA + EtBr solutions, shook well and spun briefly in a swing out rotor and upper organic phase was removed. The extraction was repeated until lower aqueous phase was completely colourless (around 5 – 6 times). Then an equal volume of Diethyl-Ether was added, solution was mixed and upper, organic phase was removed to remove rests of n- Butanol from the water phase. The Ether will remove itself during the next steps due to room temperature. DNA solutions were transferred into 50 ml tubes and diluted with 2 volumes of sterile TE. 1/10 new volume of 3 M Sodium-Acetate pH 5.2 and another 2 volumes of cold 100 % EtOH (stored at -20°C) were added, the solution was mixed and incubated on ice for 1 – 2 hours or overnight at 4°C, followed by a centrifugation step 10000 x g for 1 hour at 4°C. Supernatant was removed and washed with 10 ml cold 100 % EtOH, spun again at 10000 x g for 10 minutes and again rests of EtOH were
removed. The pellet was air-dried until transparency. DNA was dissolved under a tissue culture flow in 500 µl of sterile 10 mM Tris-HCl pH 8. Vigorous shaking was avoided! DNA solution was transferred into a 1.5 ml reaction tube, eventually followed by another spin at full speed for 5 minutes to further remove insoluble particles. Average yield: around 1 mg of pure DNA.