Nucleic Acid Extraction

2.2.1Preparation of RNase-free solutions and equipment.

Solutions used in the extraction of both RNA and DNA from cellulose baits were treated with DEPC in order to inactivate RNAses and DNAses and protect the samples from enzymatic damage. Solutions prepared in glassware were treated with the addition of DEPC to 0.05% v/v and then incubated overnight at room temperature, with rotation, and finally autoclaved to destroy the remaining DEPC. Plasticware was autoclaved before use in containers washed with RNase Zap solution (Ambion) and rinsed with DEPC treated water. Pipette exteriors and gloves were treated liberally with RNase Zap before every experiment where RNA was to be handled.

2.2.2. Nucleic acid extraction using the “Griffiths method”.

Nucleic acids were extracted from samples using the method of Griffiths et al.

(2000), which comprises mechanical bead-beating and chemical extraction, and was carried out briefly as follows. 5% (w/v) CTAB lysing buffer was prepared by combining equal quanitities of a 10% (w/v) CTAB in 0.7 M NaCl solution and 240 mM potassium phosphate buffer (pH 8). Bead-beating tubes were prepared by the addition of acid-washed glass beads (Sigma) to 2 ml screw-cap tubes. 0.5 ml 5% CTAB lysing buffer and 0.5ml Phenol:chloroform:isoamyl alcohol (25:24:1) were added to each tube. Typically, approximately 0.5g of colonised cellulose bait was processed in one tube.

Samples were subjected to bead-beating for mechanical disruption of cellular material, either in a fastprep for 30 seconds at a speed of 5 m s-1 or, as an equivalent treatment, in a MoBio Powerlyzer at a setting of 3400 RPM for 60 seconds. After bead beating, the samples were centrifuged at 17,000 x g at 4oC for 10 min. The upper aqueous phase was decanted to an equal volume of chloroform:isoamyl alcohol (24:1) and the tubes were then centrifuged as before. The upper layer was transferred to a new tube and nucleic acids precipitated with two volumes of PEG solution (30% (w/v) PEG 6000 in 1.6 M NaCl) and left to precipitate for either two hours at room temperature or overnight at 4oC. The nucleic acids were then pelleted by centrifugation at 17 000 x g for 10 minutes, and washed with ice-cold 70% ethanol in DEPC water. Residual ethanol and moisture were removed by evaporation for approx. 2 h in a fume hood or by vacuum centrifugation for 10 min at 40 oC. Pellets were resuspended in 50µl DEPC-treated water and stored at -20oC, or at -80oC for long term storage.

2.2.3. Purification of extracted nucleic acids for RNA-free DNA and DNA-free RNA. The Griffiths method co-extracts nucleic acid. In order to produce RNA-free DNA extracts, the samples were made up to 150µl with nuclease-free water, RNase A was added to 100 µg ml-1 before incubation at 37oC for 15 min. DNA-free RNA was obtained by treating RNA samples with TURBO DNA-free (Ambion) according to the manufacturer’s instructions. Briefly, 5 µl of 10x DNase buffer and 3 units of TURBO DNase were added to 50 µl of the sample and incubated at 37oC for 30 min. An additional 2 units of TURBO DNase were then added, followed by a further 30 min incubation.

In order to purify samples after nuclease treatment, 200 µl phenol:chloroform:isoamyl alcohol (25:24:1) was added. The tube was briefly vortexed and centrifuged at 10,000 x g for five min. The aqueous layer was transferred to a fresh tube, and an equal volume of chloroform:isoamyl alcohol (24:1) was added. The tube was vortexed and centrifuged as before, and the aqueous layer was again transferred to a fresh tube. The nucleic acid was precipitated with two volumes of 100% ethanol and 1/4 volume of 10M ammonium acetate, incubated for 30 min. at -80oC, and centrifuged at full speed in a microfuge for 20 min. The supernatant was discarded and the nucleic acid pellet was washed with 200 µl ice cold 70% ethanol and air-dried, or dried by vacuum centrifugation for 10 min at 40oC. The pellet was resuspended in 50µl nuclease-free water and stored at -80oC until use.

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2.2.4.Quantification and quality control of nucleic acid preparations.

A combination of methods was used. Reliable quantification was obtained using the Qubit fluorometer platform (Invitrogen). NanoDrop spectrophotometer (Thermo Scientific) readings were taken to assess purity of samples, based on the ratios of the absorbance values at 230 and 260 nm (A230/A260) and at 280 and 260

nm (A260/ A 280) for chemical and protein contamination respectively. Visualisation of

nucleic acids was achieved by agarose gel electrophoresis in order to inspect samples for signs of degradation and enzymatic damage, as evidenced by poor band integrity.

2.2.5 Extraction of genomic DNA from bacterial cells.

Genomic DNA was extracted from bacterial cell cultures according to the method of Cheng et al. (2006). Briefly, 1 ml of an overnight culture was centrifuged at full speed in a microfuge for 2 min in a microfuge tube. The supernatant was discarded and the cells were washed with 400 µl of STE buffer consisting of 100mM NaCl, 10mM Tris/HCl and 1mM EDTA at pH 8. Pellets were resuspended in TE buffer, comprising 10mM Tris/HCl and 1mM EDTA at pH 8. 100 µl Phenol was added and the tube vortexed for 60s. The tube was centrifuged at 14, 000 rpm for 5 min and the aqueous phase decanted to a fesh tube. 40 µl TE buffer and 100 µl chloroform were added, the tube mixed by inversion. Centrifugation was carried out as before and the aqueous phase was decanted to a fresh tube, quantified and stored at -20oC.