Sumisión del emperador al papado

After running the agarose gel for the appropriate length o f time the gel was placed in a suitable plastic box, covered with fresh depurination solution (250 mM HCl) and agitated on an orbital shaker until the bromophenol blue dye turned completely yellow (usually 10-20 min depending on the depth and percentage agarose o f the gel). The depurination solution was discarded and the gel was rinsed with distilled water. The gel was then covered with dénaturation solution (1.5 M NaCl, 0.5 M NaOH) and agitated for 25 min after the bromophenol blue dye had turned back to the original blue colour. This solution was discarded and the gel rinsed with distilled water. The gel was covered with

neutralisation solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.5) and agitated for 30 min.

2.20.2 Capillary Blotting.

A suitable container was part filled with 20 x SSC and a supporting platform put into position over the container. The platform was covered with three sheets o f Whatman 3MM filter paper saturated with 20 x SSC to act as a wick. The gel was placed onto the 3 MM paper making sure no air bubbles were present. The gel was surrounded with Saran Wrap (Dow Chemical Company supplied through BDH). A sheet o f Hybond N+ nylon membrane (Amersham, Little Chalfont, UK) was cut to the exact size o f the gel and

placed on top o f the gel making sure no air bubbles were present. Three sheets o f absorbant filter paper (Merck, Lutterworth, UK) cut to the size o f the gel and soaked in 10 X SSC were placed on top of the membrane, again ensuring no air bubbles were

present. A 5-7cm stack o f absorbent paper towels were placed on to the sheets o f filter paper and a suitable weight (Sigma catalogue) placed on top o f the paper towels. This was left overnight at room temperature.

2.20.3 Processing the Blot.

The blotting stack was dismantled and the membrane and gel removed together. The membrane was peeled from the gel and placed on a fresh piece o f filter paper DNA side up. The DNA side was marked with a pencil. The membrane was placed in 6 x SSC and rinsed for 1 minute with gentle agitation to remove any agarose. The membrane was then placed onto another fresh piece o f filter paper and the DNA fixed by UV cross-linking in a UV Stratalinker 1800 (Stratagene Europe, Amsterdam, Netherlands) according to the manufacturer’s instructions.

2.21 Preparation of RNA from C. difficile

2.21.1 Harvesting of C. difficile Cells

RNA was always prepared from C. difficile cells grown in modified Wilkins-Chalgren medium (Wilkins-Chalgren Anaerobe Broth 33 gf% glucose 2 g f \ sodium bicarbonate 0.2 g f \ cysteine hydrochloride (Sigma) 0.5 g f \ pyridoxal hydrochloride (Sigma) 0.01 gf* and Tris pH 7.3 1.6 gf^) the sample sizes used were 50 ml for exponential phase cells and 5 ml for stationary phase cells. Cells were cooled to approximately 4 °C in liquid

nitrogen (immersed for 2-4 sec) in a 50 ml conical tube. These were centrifuged for 3 min, 5305 g at 4 °C. The supernatant was discarded and the pellet washed once with 20 ml -20 °C acetone by centrifuging at 5305 g for 3 min 4 °C. The supernatant was discarded and the pellet frozen in liquid nitrogen. The cells were stored overnight at -70 °C with a loose cap (for the evaporation o f residual acetone) before preparing the RNA.

2.21.2 Preparation of RNA

The cells were resuspended in 400 pi o f acetic acid (2 mM, pH 4.0-4.8) and lysostaphin (24 pi o f 1 unitpl'^) was added and incubated at 37 °C for 10 min. Proteinase K (250 pg at 10 pgpl’^) was added and incubated at 37 °C for 10 min. Cell lysis was completed by adding 40 pi o f 20 % SDS and incubating at 60 °C for 10 min. Extraction was performed as follows; 400 pi hot phenol (60 °C, water saturated) was added to each tube and

vortexed briefly. The tubes were then placed in a pre-cooled ethanol/water mix (-20 °C) for 3 min. The tubes were centrifuges at 17089 g for 3 min at 4 °C and the upper phase removed to a new tube without disturbing the interphase. After extracting twice the nucleic acid was precipitated by adding 0.1 volumes of 5 M NaCl and 2.5 volumes o f -20 °C 96 % ethanol. This was incubated for 20 min at -70 °C, centrifuged at 17089 g for 25 min at room temperature and the supernatant discarded. Any residual traces o f ethanol were removed by aspiration.

The pellet was dissolved in 95 pi o f DNase Buffer (6 mM M gC^, 40 mM Tris-HCl pH 7.5) and digested with 40 U (5 pi) o f RNase-free DNase for 60 min at 37 °C. This was extracted once with phenol (equilibriated to pH 7.0) at room temperature. The sample

was centrifuged for 5 min at 17089 g at room temperature and the upper phase transferred to a new tube. This tube was stored on ice. 100 |l i1o f DNase buffer was added to the

phenol and re-extracted. The upper phases were pooled and 100 p,l o f chloroform was added, the samples were spun at 17089 g for 5 min and the upper phase transferred to a new tube. The RNA was precipitated with 96 % ethanol as described above and

redissolved in 50 pi o f DNase buffer. 5 pi o f each o f the samples were removed for gel analysis and 3 pi removed for spectrophotometric analysis at OD280/260. The RNA was

stored at -70 °C until further purification.

2.21.3 Purification of RNA

The method used for the purification o f the RNA is essentially the same as that described by Kedzierski and Porter (1991) without adding yeast. It was carried out as follows, 5 pg o f RNA was digested with 6 U o f RNase-free DNase I at 37 °C for 30 min. This was followed by adding 1 pi 0.5 M EDTA, 20 pi 3 M NaAc pH 5.2, 160 pi TE buffer and 300 pi phenol (water saturated):chloroform (1:1) and vortexing vigorously. This was spun at 17089 g for 10 mins at 4 °C and the RNA precipitated with 96 % ethanol as described above. The RNA was dissolved in DEPC treated water. cDNA production was carried out immediately with the purified RNA. Any RNA not used immediately was stored at -70 °C.