ECUACIÓN PARA LA COMBUSTIÓN COMPLETA TEÓRICA

Reagents All reagents were from Merck Ltd unless otherwise stated.

* Tris buffer, pH 8.0 with glucose and EDTA - 25 mM Tris, 50 mM glucose, 10 mM EDTA (autoclaved)

* Alkali solution - 0.2 M sodium hydroxide containing 10% (w/v) SDS * Sodium acetate solution, pH 5.2 - 3 M in distilled water

* Tris EDTA (TE) buffer, pH 8.0 - 10 mM Tris, 1 mM EDTA * Isopropanol

* Ethanol

* Tris-HCl, pH 8.0 - 10 mM Tris * Caesium chloride

* Ethidium bromide - 10 g/1 in distilled water * Butanol - water saturated

M eth o d Large scale preparations o f plasmid DNA were prepared using a modification of the alkali lysis method reported in Section 2.5.2, followed by purification by a caesium chloride density centrifugation step. Generally, 0.5 1 volumes of culture were processed.

The bacteria were first incubated at 4°C for at least 30 minutes before processing, then centrifuged at 3000 rpm for 30 minutes at 4°C and the supernatant removed. The bacteria were then resuspended in 50 mis Tris buffer with glucose and EDTA, and incubated at room temperature for 10 minutes. After this time, 100 ml of alkali solution were added and the mixture was incubated for a further 10 minutes. 75 ml potassium acetate solution were added and mixed and the flask was stored on ice for 10 minutes. The flask was then centrifuged at 3000

rpm for 20 minutes. The supernatant was removed and filtered through a gauze filter and a 0.6 volume of isopropanol was then added. After mixing and incubation at room temperature for 10 minutes, this was then centrifuged at 6000 rpm for 10 minutes. The pellet was washed once with ethanol and then dried using a vacuum desiccator.

The pellet was dissolved in TE buffer and further TE buffer was added until the total weight of the solution was 9 g. 10 g of caesium chloride were then added and mixed until solution was complete. 1 ml ethidium bromide was added and mixed and the solution was then transferred to a sealable tube (Quickseal Tube, Beckman) which was then heat sealed. The tube was the centrifuged at 55,000 rpm for 24 hours at 20°C in an 80Ti rotor in a L80 7M ultracentrifuge (both Beckman, High Wycombe, Bucks). Of the two bands of DNA located in the centre o f the gradient, the lower one represents the plasmid DNA of interest, while the upper, thinner band represents linear DNA and nicked plasmid DNA. The lower band was therefore removed by needle aspiration through the side of the centrifuge tube. The DNA was resuspended in 1.6 g/ml caesium chloride in TE buffer in a fresh sealable tube and ultracentrifuged again, using the same conditions.

The DNA was then resuspended in TE buffer and added to an equal volume of water-saturated butanol and centrifuged at 3000 rpm for 3 minutes. The aqueous phase was then added to a further volume of butanol. This was repeated until all the 'pink colour' was removed from the aqueous phase (usually 6 - 7 extraction cycles). Distilled water was then added to a volume of 9 ml and 1 ml o f sodium acetate solution was then added. Twenty ml ethanol were added and the mixture was incubated at room temperature overnight. The tube was then centrifuged at 10,000 rpm for 30 minutes at 4°C and the resultant pellet was washed in ethanol. The final DNA pellet was dried using a vacuum desiccator and resuspended in an appropriate buffer.

2.5.3 Total RNA

Reagents AU reagents were from Merck Ltd unless otherwise stated.

* Diethylpyrocarbonate (DEPC) treated water - mixed overnight at 37®C and then autoclaved.

* B mercaptoethanol (Sigma Chemical Co)

* Guanidine thiocyanate (GTC) solution, pH 7 - 4 M GTC, 0.025 M sodium citrate, 0.5% (w/v) sodium lauryl sarcosine (SLS), 0.7% (v/v) B-

mercaptoethanol.

* EDTA - 0.5 M, pH 7 in DEPC treated water

* Caesium chloride solution - 240 g Caesium chloride and 5 ml EDTA was made to 250 ml with DEPC treated water.. This was incubated overnight at 37°C overnight and then autoclaved.

* Sodium acetate - 2M * Ethanol

* Sodium acetate solution - 0.3 M sodium acetate, pH 4 containing 0.1% (w/v) SDS and 10 mM EDTA

* Precipitation solution - 3 volumes of sodium acetate solution added to 8 volumes of ethanol (obtained from a previously unopened bottle). Prepared immediately before use.

M ethod Isolation of RNA was performed according to the method of Chirgwin et al (1979). Care was taken at all times to minimise the risk o f RNase contamination. All glassware was DEPC treated overnight and then autoclaved, and aU solutions were prepared in DEPC treated water.

Cells previously stored in GTC at 4 - 20®C (6 ml of GTC / 10^ cells), had their DNA sheared by passing the GTC cell lysate six times through a 23 gauge needle (Terumo, Leuven, Belgium). The GTC lysate was then carefully layered onto 5 ml of caesium chloride solution cushion in ultracentrifuge tubes (14 x 89 mm, Beckman). The ultracentrifuge tubes were then centrifuged at 27 000 rpm for

22 hours at 20°C in a SW41 rotor and L80 7M ultracentrifuge (both Beckman). The resulting supernatant was gently aspirated with a drawn out pasteur pipette, being careful not to disturb the pellet of RNA. The ultracentrifuge tube containing the pellet was inverted and residual GTC allowed to drain. The cylindrical top of the ultracentrifuge tube was cut off with a hot scalpel and the RNA pellet at the base of each tube was precipitated by addition of 200 |il precipitation solution and incubation at room temperature for 1 hour. The resulting opaque RNA pellet was carefully transferred to a 1.5 ml microfuge tube containing 900 |il o f the precipitation solution. This was allowed to further precipitate at 4°C for 24 hours and finally pelleted by centrifugation at 10,0(X) x g for 20 minutes. The pellet was washed twice in 70% ethanol and then dissolved in 50 - 200 |il of DEPC water.