2.2.1 - Mini preps
Mini preps were prepared as described by Grosveld et al (1981).
10 ml of L Broth (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, pH 7.2) containing 0.1 mg/ml ampicillin was inoculated, either from a bacterial colony or a
glycerol stock and incubated overnight at 37®C in a shaking incubator.
The culture was centrifuged at 3,000 r.p.m for 20 minutes at 40C in a Beckman J-6B centrifuge. The resulting pellet was resuspended in 200 pi of Tris-glucose, to which 400 pi of 1% SDS, 0.2M NaoH were added at room temperature. This was left at room temperature for 10 minutes in order to lyse the bacterial cells. Next 200 ml of 3M KAC was added, the solution was vortexed and microfuged at 13,000 r.p.m for 10 minutes.
The aqueous phase was removed and 0.6 times the volume of iso-propanol added, they
were vortexed and centrifuged as before. The pellet was washed with 70% (v/v) ethanol and microfuged for another 10 minutes. The ethanol was removed and the pellet freeze dried, then resuspended in 50 |il of TE (lOmM Tris-Cl, ImM EDTA, pH 8.0). The DNA was RNA treated with 1 |il of 10 mg/ml RNAse A at 37®C for 1 hour. The DNA was then cleaned by phenol/chloroform extraction and resuspended in 50 |il of TE.
2.2.2 - Phenol/chloroform extraction of nucleic acids
The DNA was cleaned with 50:49:1 (v/v) phenol/chloroform/isoamyl alcohol and vortexed. This was followed by microfuging at 13,000 r.p.m for 5 minutes. The lower phenol layer was discarded and an equal volume of equilibrated chloroform added and again vortexed and microfuged for 20 seconds. The lower organic layer was discarded
and the concentration of anionic salt in the DNA solution adjusted to the appropriate levels, to a final concentration of 0.3M sodium acetate, pH 5.2. This was followed by the addition of 2.5 times the volume of ethanol, then vortexed and incubated at -20®C for 2 hours. The ethanol/DNA was microfuged for 15 minutes and the resulting pellet washed with 70% (v/v) ethanol, again spun for 5 minutes, the pellet was freeze dried and resuspended in an appropriate volume of TE.
2.2.3 - Electrophoretic separation of DNA fragments
Restriction digests were set up to check the DNA obtained from the mini preps. The
digest was set up using a maximum of 1 |ig of DNA to which 2|il of the relevant buffer for the enzyme being used (manufacturers recommendation) was added, the volume was then made up to 19 |il with water and 1 unit of enzyme added. This was then incubated for 1 hour at 37^0.
An agarose gel was prepared of 1% (w/v) agarose (Sigma, Poole, Dorset) in 1 x TBE
(Tris-Borate-EDTA - lOxTBE, 1 M Tris-Cl, IM Boric acid, 20 mM EDTA, pH 8.35) and 0.5 M-g/ml of ethidium bromide. The running buffer was comprised of 1 x TBE. 10
|il of the enzyme digest was taken and 1 }il of loading dye added (0.25% (w/v) bromophenol blue, 25mM EDTA, 50% (v/v) glycerol), then loaded on to the gel. The gel was run at 75 volts and visualised on a short wave ultraviolet transilluminator (254 nm) and photographed with Polaroid type 667 film (Polaroid, UK, St.Albans, Herts).
2.2.4 - Maxi prep: Lithium chloride method
The protocol used is an unpublished procedure of R. Treisman.
A conical flask containing 500 ml of L-broth was prepared and autoclaved, 500 |xl of (0.1 mg/ml) ampicillin was added and it was inoculated with bacterial glycerol stocks of the DNA required. This was incubated in a 37°C shaking incubator overnight. The next day the culture was centrifuged at 3,000 r.p.m in a Beckman J-6B centrifuge for 30 minutes at 4^0 and the bacterial pellet was resuspended in 18 ml of solution 1 (50 mM glucose, 25 mM Tris-Cl, pH 8.0, 10 mM EDTA). Next 40 ml of solution 2 was added (0.2 M sodium hydroxide, 1% (w/v) SDS) and thorough mixing ensured by inverting the centrifuge bottle several times, this was then incubated at room temperature for 10 minutes. 20 ml of solution 3 was then added (60% (v/v) 5M potassium acetate, 11.5% (v/v) glacial acetic acid). The mixture was then shaken and stored on ice for 10 minutes, to allow a precipitate to form. This was then centrifuged at 4,000 r.p.m in a Beckman J-
6B centrifuge (with the brake switched off), 15 minutes, 4^C, a tight pellet formed consisting of the bacterial debris. The supernatant was then filtered through four layers of cheesecloth into a fresh 250 ml Beckman centrifuge bucket and 0.6 volume of iso propanol added, mixed well and incubated at room temperature for 10 minutes. This was then centrifuged at 5,000 r.p.m, 15 minutes, 4°C in a Sorval GS3 rotor, in a Sorval RC 5B centrifuge, and the resulting pellet carefully washed with 70% (v/v) ethanol, air dried and resuspended in 3 ml of TE.
The plasmid DNA was then purified. The DNA was transferred to a 15 ml corvex tube
and 3 ml of ice cold 5M lithium chloride added to precipitate the high molecular weight RNA, mixed well and centrifuged at 10,000 r.p.m in a Sorval SS34 rotor, in a Sorval RC 5B centrifuge for 10 minutes, 4^0. The supernatant was carefully poured away and
discarded and the pellet washed with 70% (vA^) ethanol. The ethanol was poured off and the pellet left to dry at room temperature for a few minutes, it was then resuspended in 500 p.1 of TE containing DNase-free pancreatic RNAse A (20 |Xg/ml), this was transferred to an eppendorf and incubated at room temperature for 30 minutes. To this 500 pi of 1.6M sodium chloride, 13% (w/v) polyethylene glycol (PEG 8000) was
added. This was mixed well and then microfuged at 12,000 r.p.m for 5 minutes, 4®C. The supernatant was discarded and the pellet resuspended in 400 pi of TE, pH 8. This was then cleaned by phenol/chloroform extraction (see section 2.2.2). The aqueous phase was removed to a fresh eppendorf, to which 100 pi of lOM sodium acetate was added and 2.5 times the volume of ethanol, vortexed and incubated at room temperature for 10 minutes. This was then microfuged for 5 minutes at 12,000 r.p.m, 4°C and the pellet was washed carefully with 70% (v/v) ethanol. The pellet and 70% (v/v) ethanol were then microfuged for 2 minutes at 12,000 r.p.m, 4°C. The 70% (v/v) ethanol was then removed and the DNA pellet allowed to air dry, before resuspending it in 500 pi of TE, pH 8.0. The optical density (O.D) at 260 and 280 nm was then measured to ascertain the yield of DNA. (1 O.D of DNA at 260 = 50 pg/ml, the O.D 260/280 ratio for
DNA = 1.8).