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2.6.1. Small scale plasmid preparations (mini-preps)

5ml aliquots of LB containing the appropriate antibiotic(s) were inoculated with single bacterial colonies containing the desired plasmid constructs These were incubated at 37°C overnight, shaking. 1.5ml of the cultures were transferred into Eppendorf tubes and centrifuged for 1 min. The remainder of the cultures were stored at 4°C. The medium was removed by aspiration and the pellets resuspended in an ice cold solution of GTE. The tubes were left at room temperature for 5 mins. The cells were lysed by addition of 200|ll of a freshly prepared solution of 0.2M NaOH/1% SDS. The tubes were mixed by rapid inversion and stored on ice for 5 mins. The bacterial chromosomal DNA was precipitated by adding 150|il ice cold potassium acetate (KAc) pH 4.8, vortexing and incubating on ice for 5 mins. The chromosomal DNA was pelleted by centrifuging at room temperature for 5 mins. The supernatants were transferred to fresh tubes containing an equal volume of PCIA. These were vortexed and centrifuged for 2 mins. The supernatants containing plasmid DNA were transferred to fresh tubes and precipitated by addition of 2 volumes of absolute ethanol. The tubes were left at room temperature for 5 mins and the DNA pelleted by centrifugation for 10 mins. The supernatants were decanted and the pellets washed in 70% ethanol. The pellets were then dried for 10 mins in a vacuum dessicator and resuspended in 30pl TE pH 8.0 containing 20|ig/ml DNAse-free RNAse. 2-5p.l were analysed by restriction endonuclease digestion.

2.6.2. Large scale plasmid preparations (maxi preps)

5ml of LB containing the appropriate antibiotics was inoculated with a single bacterial colony and grown during the day at 37°C, shaking. This culture was used to inoculate 400ml LB in a 1 litre flask and further grown at 37°C, shaking, overnight. The bacterial cells were harvested in a 500ml centrifuge bottle by spinning at 6(XX)rpm for 10 mins at 4°C using a Beckman JAIO rotor. The pellet was resuspended in 10ml of GTE and left at room tem perature for 5 mins. The suspension was transferred to a 100ml glass flask on ice, and 20m l 0.2M N aOH/0.1% SDS added. The suspension was mixed by swirling and left for 5 mins on ice to lyse the cells. 10ml o f 5M KAc pH 4.8 was then added, mixed by sharp inversion and incubated on ice for 15 mins. The bacterial DNA and cell debris were precipitated by centrifugation for 15 mins, 3(XX)rpm, 4°C in a Heraeus M inifuge T. The supernatant was strained through gauze into a 200ml glass bottle and the DNA precipitated by the addition of 0.6 volumes of isopropanol. A fter incubation at room tem perature for 30 m ins, the DNA was pelleted by centrifugation at 2000rpm for 15 mins in a Beckman J-6B. The supernatant was discarded and the pellet allowed to air dry. The crude plasmid DNA was then resuspended in 3ml TE pH 8.0. This was added to 4.4g caesium chloride (CsCl) in a sterilin tube along with TE pH 8.0 until a total of 4g o f liquid had been obtained. 400|il of lOmg/ml ethidium bromide was added and the density of the solution adjusted to 1.5-1.6g/ml. The solution was centrifuged at 3(X)0rpm for 10 mins at room temperature in a Heraeus minifuge T to precipitate bacterial proteins. The clear supernatant was transferred to a Beckman polyallomer Quick Seal centrifuge tube and the tube filled with Ig/ml CsCl in TE pH 8.0. The tubes were sealed and centrifuged at 58(X)Orpm in an ultracentrifuge, 15°C for 18 hrs.

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The plasmid DNA band in the resulting CsCl gradient was visualised under

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ultraviolet (uv) light. The top o f the tube was pierced with a 25g needle and the plasmid DNA extracted with a 19g needle in 0.5-0.7 mis solution. Care was taken to avoid both the RNA band down the side of the tube and the upper nicked plasm id DNA band. The ethidium bromide was rem oved from the solution by repeated butan-2-ol extractions. The volume was increased three fold by addition o f TE pH 8.0, two volum es o f absolute ethanol were added and the DNA precipitated by centrifugation at 4000rpm , room tem perature in a H eraeus minifuge T. The supernatant was decanted and the pellet resuspended in 400pl TE pH 8.0. This was transferred to an Eppendorf tube and precipitated by addition of

40|il NaAc pH 5.2 and two volumes of absolute ethanol. The DNA was pelleted by centrifugation for 10 mins in a microfuge, room temperature. Two further precipitations were performed before washing the pellet in 70% ethanol and final resuspension in 400|xl TE pH 8.0 containing 20|xg/ml DNase-free RNase. The yield was determined by measuring the absorbance of an aliquot at OD2 6onm.

2.6.3. Small scale PI preparations

A single bacterial colony was inoculated into 10ml 2YT medium containing 25pg/|xl kanamycin and grown overnight, shaking, at 37°C. The cells were spun down at 2000rpm for 10 mins in a Beckman centrifuge and the pellet resuspended in SOOpl GTE. The solution was transferred to an Eppendorf tube and incubated on ice for 5 mins. 600|xl of a freshly prepared solution of 0.2M NaOH/1% SDS was added, mixed by inversion, and the tube incubated on ice for 5 mins. The bacterial DNA was pelleted by centrifugation for 10 mins at room temperature in a microfuge. The supernatant was transferred to a fresh Eppendorf tube, the tube filled with isopropanol, and the PI DNA left at room temperature for 30 mins to precipitate. The tubes were spun in a microfuge for 15 mins and the supernatant decanted. The pellet was air dried and resuspended in lOOjxl TE pH 8.0. The PI DNA pellet was purified as follows. Ammonium acetate was added to a final concentration of 2.5M, the solution incubated on ice for 10 mins, and the debris pelleted by centrifugation for 10 mins in a microfuge. The supernatant was transferred to a fresh Eppendorf tube and extracted once with phenol, once with PCIA and once with CIA. The aqueous phase was precipitated by the addition of NaAc pH 5.2 to a final concentration of 2.5M and two volumes of ethanol. The DNA was precipitated by centrifugation for 10 mins at room temperature in a microfuge. The supernatant was decanted, the pellet dried in a vacuum dessicator and resuspended in 20|xl TE pH 8.0.