Large scale (maxi prep)

A single colony of transformed E.coli cells was inoculated into 5-10ml of LB, containing the appropriate antibiotic. The culture was incubated at 37°C with vigorous agitation for 6 - 8 hours until turbid. It was then transferred into 250ml of LB,

containing the appropriate antibiotic and left shaking (250rpm) for 12-16 hours in the 37°C incubator. Cells were pelleted by centrifugation (6,000rpm in a Sorvall centrifuge) at 4°C for 10 min. The pellet was completely resuspended in 10ml Dish 1 (see section 2.3.1) by vortexing until no cell clumps were visible and placed on ice. Twenty ml of freshly made Dish 11 (see section 2.3.1) were then added drop wise and the mixture was incubated on ice for no more than 10 minutes. Finally, 10ml of ice- cold Dish 111 (see section 2.3.1) were added, mixed by swirling and left on ice for at least 30 min. After centrifugation (10,000rpm at 4°C for 10 min) the supernatant was filtered through an absorbent cotton gauze (BP type 13 light by Smith & Nephew Textiles Ltd., UK) into a clean tube. DNA was precipitated by adding two volumes of 100% ethanol and recovered by centrifugation at 10,000rpm for 10 min at RT. The supernatant was discarded and the pellet washed in 70% (v/v) ethanol, centrifuged again and air-dried for approximately 20 min at 37°C before resuspending in 4ml TE buffer.

T o purify the plasm id D N A , ethidium brom ide-caesium chloride (E tB r-C sC l) density gradient centrifugation w as then performed. A den sity gradient is produced by centrifugation o f a CsCl solution at a very high speed. M acro m olecu les present in the C sCl solution form bands at distinct points in the gradient dep en din g on their buoyant d en sity (Figure 2.1a). D en sity gradient centrifugation m the presence o f EtBr is used to separate supercoiled D N A from non-supercoiled m olecu les. EtBr binds D N A causing partial unw inding o f the helix which results in a d ecrease about 0.125g/cm ^ in buoyant density for linear D N A . Supercoiled D N A , h avin g no free ends, can bind o n ly a lim ited amount o f EtBr causing a decrease in buoyant density o f about 0 .0 8 5 g /c m \ W hen the cell lysate is subjected to the ab ove procedure, plasm id D N A is separated from linear bacterial D N A (Figure 2.1b).

Protein Protein Protein Linear DNA Increasing CsCl density DNA Supercoiled DNA RNA RNA RNA

F ig u re 2.1 E th id iu m b ro m id e-ca esiu m ch lorid e (E tB r-C sC l) d en sity grad ien t c en trifu g a tio n , a) S eparation o f m acrom olecules in a density g radient, b) D ensity gradient in the p resence o f E tB r separates supercoiled (plasm id) D N A from linear (bacterial) D N A , and c) R em oval o f supercoiled (p lasm id ) DNA.

S ubsequently, 4 .8 g o f CsCl w ere added to the 4m l D N A su spension, the tube was gen tly inverted to d isso lv e the C sCl before adding 5 0 0 p g /m l EtBr. The mixture was then centrifuged (3,500rpm in a D en ley B S 4 0 0 centrifu ge) for 10 m in, and the clear red solution was transfeired into a Q uick-Seal tube (B eckm an Ltd., U K ). The tubes

were balanced and spun overnight (12-16 hours) at RT in a Beckman L-80 ultra­ centrifuge (NVT 90 rotor) at 70,000rpm.

Next day, the plasmid DNA band was removed by puncturing the side of the Quick- Seal tube and withdrawing it into a syringe (Figure 2.1c). The EtBr bound to plasmid DNA was extracted at least three times with equal volumes of isoamyl alcohol saturated with water. The mixture was spun each time at 3,000rpm for 3 min (Denley BS400 centrifuge) and the aqueous lower (DNA-containing) phase was removed. Two volumes of water were then added to reduce CsCl concentration and the DNA was precipitated with two volumes of ice-cold 100% ethanol at -20°C for at least 15 min.

The DNA pellet was recovered by centrifugation in a Sorvall centrifuge (10,000rpm) for 15 min at 4°C, resuspended in 1ml of water and transferred into an 1.5ml eppendorf. Three organic extractions, as described in section 2.7, were performed: two with phenol:chloroform:isoamyl alcohol (25:24:1) and one with chloroform : isoamyl alcohol (24:1). Each time, the aqueous upper (DNA-containing) phase was carefully removed into a clean 1.5ml eppendorf. DNA was precipitated, washed and dissolved in water (see section 2.7).

2.9 Measurement of DNA concentration

DNA concentrations were measured by UV absorbance spectrophotometry using a DU650 series spectrophotometer (Beckman Ltd., UK). Genomic or plasmid DNA was diluted (1/100) in TE buffer and 500pl were added to a microcuvette. The spectrophotometer was blanked with TE buffer, the density of the sample was measured at 260nm and at 280nm and the DNA concentration was calculated as follows:

OD260 X 50jLig/ml X dilution factor

One CD unit at 260nm is defined as 50pg/ml for double stranded DNA. The 260nm/280nm ratio represents the purity of the sample. In high quality DNA, free of contaminants such as protein or phenol, the 260nm/280nm ratio ranges from 1.8-2.0. Concentration and quality of DNA were also verified by conventional gel electrophoresis (see section 2.11).