CAPACIDAD DE CARGA (qu)

2.3.1 Mini-preps (1-2 ml o f a 5 ml overnight culture)

2.2.1 a) Rapid method (TENS)

Single colonies were inoculated into a 5 ml culture of LB media containing 50 |ig/ml ampicillin, using aseptic technique and grown overnight at 37 °C, 225 rpm. 1.5 ml o f the overnight cultures were centrifuged at 13,000 rpm for 10 seconds. The supernatant was discarded in one shake and the pellet resuspended in the remaining 50-100 pi o f LB. 300 pi of TENS (10 mM Tris pH 7.5, 1 mM EDTA pH 7-8, O.lM NaOH, 0.5% SDS) was added and vortexed for 2-5 seconds (the samples were stored on ice if left for more than 10 seconds before the next step). 150 pi of 3M sodium acetate pH 5.2 was added and the samples vortexed for a further 2-5 seconds. The samples were then centrifuged at 13,000 rpm for 2 min and the supernatant transferred to a fresh tube. 900 pi of absolute ethanol was added and centrifuged at 13,000 rpm for 6 min. The pellet was washed twice with 70% ethanol then dried at 37 °C for 5-10 min and resuspended in

100 pi TE pH 8 (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0).

2.3.1 b) Qiagen QIAprep Spin Plasmid Kit

This procedure was used when a cleaner DNA sample was required e.g. for sequencing. 2 ml of the overnight cultures were centrifuged at 13,000 rpm for 2 min. The supernatant was discarded and the pellet was resuspended in 250 pi of resuspension buffer P i (100 pg/ml RNAse A, 50 mM Tris/HCl, 10 mM EDTA pH 8.0). 250 pi of lysis buffer P2 (200 mM NaOH, 1% SDS) was added and the samples were inverted to mix and incubated at room temperature for 5 min. 350 pi o f chilled buffer N3 (containing guanidine hydrochloride) was added, and the samples mixed immediately

by gentle inversion. The samples were incubated on ice for 5 min. The samples were then centrifuged at 13,000 rpm for 10 min. A QIAprep spin column was placed in a 2 ml microcentrifuge tube and the supernatant from the samples was added to the column. These were centrifuged at 13,000 rpm for 1 minute, and the flow-through fraction was drained from the tube. An optional wash with buffer PB was carried out when using E.coli strains with high nuclease activity, by adding 0.5 ml of buffer PB to the column and centrifuging at 13,000 rpm for 1 minute. The column was washed once with 0.75 ml of buffer PE and centrifuged for 1 minute. The column was centrifuged for a further minute to remove residual wash buffer. The column was placed in a clean 1.5 ml microcentrifuge tube and the DNA was eluted by the addition of 100 pi water followed by centrifugation at 13,000 rpm for 30 seconds. The DNA was stored at -2 0 °C.

2.3.2 Qiagen maxi-prep

Used for the preparation of up to 500 pg of DNA used for in vitro studies.

A single bacterial colony was used to inoculate a starter culture o f 5 ml of LB media containing ampicillin and grown for approximately 8 hours at 37 ®C, 225 rpm. 250 pi of the starter culture was inoculated into 250 ml of LB media containing ampicillin and grown overnight at 37 °C, 225 rpm. The following day the culture was pelleted by centrifugation at 6,000 x g, 4 °C for 10 min, the supernatant was removed and the protocol in the Qiagen® plasmid handbook was followed: the pellet was resuspended in 10 ml buffer PI (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 100 pg/ml RNAse A), leaving no cell clumps, 10 ml of Buffer P2 (200 mM NaOH, 1% SDS) was added and mixed gently, the solution was then incubated at room temperature for 5 min. 10 ml o f

chilled buffer P3 (3.0 M potassium acetate, pH 5.5) was added, the solution mixed immediately but gently and incubated on ice for 20 min. Following incubation, the sample was centrifuged at 20,000 x g for 30 min at 4 °C and the supernatant promptly removed; if the supernatant was not clear a second shorter centrifugation was performed or the supernatant was filtered over a pre-wetted, folded filter paper. A QIAGEN-tip 500 was equilibrated by applying 10 ml of Buffer QBT (750 mM NaCl; 50 mM MOPS, pH 7.0; 15% ethanol; 0.15% Triton X-100) and allowing the column to empty by gravity flow. The clear supernatant from the previous step was applied to the QIAGEN- tip 500 and allowed to enter the resin by gravity flow. The QIAGEN-tip 500 was washed twice with 30 ml of Buffer QC [1.0 M NaCl; 50 mM (3-[A- Morpbolinojpropanesulfonic acid buffered saline (MOPS), pH 7.0; 15% ethanol) and the DNA eluted from the tip by adding 15 ml of Buffer QF (1.25 M NaCl; 50 mM Tris- HCl, pH 8.5; 15% ethanol]. The DNA was precipitated with 10.5 ml o f isopropanol (equilibrated to room temperature) and was centrifuged immediately at 15,000 x g for 30 min at 4°C. The supernatant was carefully removed to avoid disturbance o f the pelleted DNA. The DNA was then washed with 5 ml o f 70% ethanol, air dried for 5 min and dissolved in approximately 300pl of sterile water. The DNA was stored at -2 0 °C.

2.3.3 Qiagen mega-preps

Used for the preparation of up to 2.5 mg of DNA for use in in vivo studies. A starter culture was prepared as described in Section 2.3.2. For each mega prep 2 x 250 ml of LB media containing ampicillin were inoculated with 250 pi o f the starter culture and grown overnight at 37°C, 225 rpm. The following day the bacteria were pelleted as for the maxi prep and DNA prepared according to the protocol in the Qiagen® plasmid

handbook, using the solutions described in the maxi prep protocol (Section 2.3.2). The bacterial pellet was resuspended in 50 ml of Buffer P I, 50 ml of Buffer P2 was added, mixed gently and incubated at room temperature for 5 min. 50 ml of chilled Buffer P3 was added, mixed immediately but gently and incubated on ice for 30 min. The sample was mixed again and centrifuged at 20,000 x g for 30 min at 4 °C and the supernatant removed promptly. If the supernatant was clear then the next step was carried out; if not a further centrifugation was performed or the sample was filtered over a pre-wetted, folded filter paper. A QIAGEN-tip 2500 was equilibrated by applying 35 ml of Buffer QBT to the column and allowing it to empty by gravity flow. The clear supernatant was applied and allowed to enter the resin by gravity flow. The QIAGEN-tip 2500 was washed 4 x with 50 ml of Buffer QC and the DNA eluted with 35 ml of Buffer QF. The DNA was precipitated immediately with 24.5 ml isopropanol (equilibrated to room temperature) and centrifuged at 15,000 x g for 30 min at 4 °C. The supernatant was carefully removed and the DNA washed with 7 ml of 70% ethanol, air dried for 10 min and dissolved in sterile water for injection. The DNA was stored at -20 °C.