ORGANISMOS INTERNACIONALES DE CARÁCTER FINANCIERO.

Plasmid DNA was amplified by growth o f transformed bacteria in liquid culture (section 2.2.1.2). Plasmid DNA was prepared on a small ‘m ini-prep’ scale from a 10ml culture (section 2.2.2.1) or on a large scale from a 500ml culture (section 2.2.2.2).

2.2.2.1 ‘M iniprep’ preparation o f plasmid DNA

Small scale purification o f plasmid DNA from bacterial culture was performed using the Magic Minipreps™ DNA Purification System (Promega), following the manufacturer’s instructions. The protocol involves isolation o f plasmid DNA by alkaline lysis and subsequent purification by binding to a silica-based matrix. Briefly, 1ml o f bacterial culture grown as described in section 2.2.1.2 was transferred to a 1.5ml tube and centrifuged at 13000rpm for 5mins.. The bacterial pellet was resuspended in 200pl Cell Resuspension Solution. Cells were lysed by the addition o f 200pl Cell Lysis Solution with mixing by repeated inversion. The lysate was neutralized by the addition o f 200pl Cell Neutralization Solution with mixing by repeated inversion. The solution was centrifuged at 13000rpm for 5mins.. The supernatant was transferred to a fresh tube and mixed with 1ml Magic Miniprep DNA Purification Resin. The DNA-resin solution was loaded onto a Magic Minipreps mini-column through a 2ml syringe. The column was washed with 2ml Column Wash Solution, transferred to a 1.5 ml tube and centrifuged at 13000rpm for 1 minute. The column was transferred to a fresh 1.5 ml tube and 50pl TE pH 7.6 pre-heated to 65-70°C was applied. Plasmid DNA was eluted from the column by centrifugation at 13000rpm for 1 minute. Plasmid DNA was subjected to analysis by restriction enzyme digestion (section 2.2.3.3) and agarose gel electrophoresis (section 2.2.3.4). Magic Miniprep-purified plasmid DNA was stored at -20°C in TE, pH 7.6.

2.2.2.2 Large scale preparation o f plasmid DNA

Bacteria picked from a freshly-grown colony (section 2.2.1.1) was used to inoculate 1ml of LB or TB growth media (377). The culture was incubated in the presence o f the appropriate antibiotic for 12-16hrs. at 37°C with shaking at 300 cycles/min., and used to inoculate 10ml LB (small scale plasmid preparation) or 500ml TB (large-scale plasmid preparation). The culture was incubated in the presence o f the appropriate antibiotic for

12-16hrs. at 37°C with shaking at 300 cycles/min..

Harvest and lysis o f bacteria

The culture was subjected to alkali lysis following a modification o f the protocols o f Bimboim and Doly (378) and Ish-Horowicz and Burke (379). Briefly, 500ml bacterial culture was harvested by centrifugation at 5000rpm, 4°C for 20mins., the bacterial pellet resuspended in Solution I and lysed in Solution II at room temperature for 10 mins.. Chromosomal DNA, high molecular weight RNA and potassium-SDS-protein- membrane complexes were precipitated by the addition o f ice-cold Solution III. The solution was incubated on ice for 10 mins. and the precipitate pelleted by centrifugation at 4000rpm, 4°C for 15mins.. Nucleic acid in the resulting supernatant was precipitated by the addition o f 0.6 volumes o f isopropanol (BDH-Merck, Lutterworth, Leicestershire, U.K.) and recovered by centrifugation at 5000rpm, room temperature for 15mins.. The pellet was washed in 70% ethanol, recovered by centrifugation at 2500rpm for 5mins., air-dried and resuspended in TE pH 7.6. Plasmid DNA was purified by polyethylene glycol (PEG) precipitation or by equilibrium centrifugation in a cesium chloride (CsCl) - ethidium bromide (EtBr) gradient followed by dialysis (380) (see below).

Purification o f plasmid DNA by polyethylene glycol (PEG) precipitation

High molecular weight RNA was precipitated from the nucleic acid solution by addition o f ice-cold 5M LiCl and centrifugation at lOOOOrpm, 4°C for lOmins.. Nucleic acid in the resulting supernatant was precipitated by the addition o f an equal volume o f isopropanol. The solution was incubated on ice for lOmins. and the nucleic acid recovered by centrifugation at 13000rpm, for lOmins.. The pellet was washed with 70% ethanol, centrifuged at 7500rpm for 5mins., air-dried and resuspended in TE pH 7.6 containing 20pg/ml DNAase I-ffee pancreatic RNAase A. After incubation at room temperature for 30mins., plasmid DNA in the solution was precipitated by the addition

o f 13% PEG, 1.6M NaCl and recovered by centrifugation at 4000rpm, 4°C for 5mins.. Purification was performed by phenol: chloroform: isoamyl alcohol (lAA) extraction (section 2.2.2.S). DNA was concentrated by ethanol precipitation (section 2.2.2.9). Plasmid DNA was resuspended in TE pH 7.6 and stored at -20°C. The concentration and purity o f plasmid DNA was determined by spectophotometry (section 2.2.3.1). Plasmid integrity was confirmed by restriction enzyme digestion analysis (section

2.2.3.3).

Purification o f plasmid DNA by equilibrium centrifugation

CsCl was added to nucleic acid solution at Ig/ml, and dissolved by incubation at 30°C. EtBr was added to a final concentration o f 0.8mg/ml. The CsCl-EtBr-DNA solution was transferred to a Beckman Quick-Seal® centrifuge tube, and the tube heat-sealed. The density gradient was spun at 60000rpm, 20°C for 24hrs.(Beckman ultracentrifuge). The lower o f the two visible bands o f DNA, which contains closed circular plasmid DNA, was collected through a 21-gauge hypodermic needle. EtBr was removed from the DNA solution by repeated extraction with NaCl-saturated isopropanol, and concentrated by ethanol precipitation (section 2.2.2.9). Plasmid DNA was purified by dialysis. Ultra Pure^'^ dialysis tubing (% inch diameter, Gibco BRL) was prepared by thorough washing with ddH]0. A hole was cut in the lid o f a 1.5ml tube containing plasmid DNA resuspended in 1.2ml TE pH 7.6. Dialysis tubing was stretched across the neck o f the tube and the lid closed, avoiding air bubbles at the interface. Plasmid DNA was dialysed at 4°C for 14-16hrs. with stirring, against a minimum volume o f 500ml TE pH 7.6. TE buffer was changed as often as possible. Purified plasmid DNA was concentrated by ethanol precipiation (section 2.2.2.9). The concentration and purity o f plasmid DNA was quantified by spectophotometry (section 2.2.3.1), and integrity confirmed by restriction enzyme digestion analysis (section 2.2.3.3). Plasmid DNA was stored at -20°C in TE pH 7.6.

2.2.2.3 Preparation o f genomic DNA from mammalian cell lines

Genomic DNA was prepared by a modification o f the procedure o f Blin and Stafford (381). Pelleted cells were washed in PBS (Oxoid Unipath Limited, Basingstoke, U.K.) and lysed in 0.3M NaCl, lOmM Tris pH 7.6, Im M EDTA, 0.5% SDS, 0.2mg/ml

proteinase K by incubation at 56°C for 1 hour. DNA was purified by phenol: chloroform: lAA alcohol extraction (section 2.2.2.8) and concentrated by ethanol precipitation (section 2.2.2.9). The resulting pellet was resuspended in TE pH 7.6 and stored at 4°C.

2 2.2.4 Preparation of total RNA from mammalian cell lines

Solutions used in RNA manipulation were treated with the RNAase inhibitor diethyl pyrocarbonate (DEPC) wherever possible. Solutions containing 0.1% DEPC were left at room temperature for 12-16hrs. and autoclaved (section 2.1.2). Glassware was soaked in DEPC-ddH20 for 2hrs., rinsed several times with ddH20, and autoclaved. Tips and plasticware were autoclaved. Electrophoresis tanks were cleaned with detergent, rinsed in ddH2Û, dried with ethanol, soaked in 3% H2O2 and rinsed thoroughly in DEPC-treated

ddH20. Gloves were worn at all times. RNA extraction was performed using Gilson pipettes not used for DNA manipulation. Total RNA was extracted from approx. 5x10^ cells under RNAase-free conditions. Briefly, cells were lysed in 0.2ml RNAzoF'^ B (Biogenesis Limited, Bournemouth, U.K.) per 10^ cells. RNA was chloroform extracted by the addition o f chloroform to 10% v/v. Samples were shaken vigorously for 15 seconds, incubated on ice or at 4°C for 5mins. and centrifuged at ISOOOrpm, 4°C for 15mins.. The colourless aqueous upper phase containing RNA was removed, and RNA precipitated by the addition o f an equal volume o f isopropanol, incubation at 4°C for 15mins. and centrifugation at ISOOOrpm, 4°C for 15mins.. The RNA pellet was washed in 75% ethanol, and re-pelleted by centrifugation at 7500rpm, 4°C for 15mins.. The pellet was dried under vacuum for 10-15mins., and dissolved in or ImM EDTA-DEPC pH7. The integrity o f extracted RNA was determined by spectrophotometry (section 2.2.3.1) and agarose gel electrophoresis (section 2.2.3.4).