CONTENIDO DE LA PROPUESTA

2.2.2.1 Standard Polymerase Chain Reaction (PCR)

Standard PCR amplifications were carried out in a final volume of 50µl in 0.5ml microcentrifuge tubes using a Primus 96 advanced Gradient Innovative PCR Technology (PeQLab). The reaction mixture usually included 50ng of template DNA, 100ng of each primer, 0.25µM of each dNTP, 5µl of 10X PCR Buffer, 1.5mM of MgCl2 and 2 units of Taq polymerase (Invitrogen). The mixture was incubated at

94°C for 1 min and subjected to 40 PCR cycles as follows: denaturing at 94°C for 1 min, annealing at 55-60°C for 1 min and extending at 72°C for 1 min/kb of amplicon. The annealing temperature was adjusted depending on the GC content of the specific primers used in order to reduce non-specific amplification in some individual PCR reactions.

2.2.2.2 Site-Direct Mutagenesis PCR

All mutagenesis PCR was performed with Platinum Pfx DNA polymerase

(Invitrogen) to ensure higher fidelity. According to the manufacture’s instruction, MgSO4 was used as a source of magnesium. The reaction mixture (typically 50µl)

5µl of 10X PCR Buffer, 1.5-3mM of MgSO4 and 2 units of Platinum Pfx DNA

polymerase was incubated at 94°C for 1 min followed by 30 cycles of 94°C for 1 min, 55-58°C for 1 min and 68°C for 1 min/kb of amplicon. The annealing temperature was adjusted depending on the GC content of the specific primers used and the final concentration of magnesium was adjusted in some individual PCR reactions for optimization. The mutagenesis PCR product was digested with Dpn I to remove methylated template before ligating into the appropriate vectors.

2.2.2.3 Restriction Enzyme Digest

Typically up to 1µg of plasmid DNA or PCR product was digested in a total reaction volume of 50µl. Restriction enzymes were used with recommended reaction buffers according to the manufacturer’s instructions. In reactions requiring multiple

restriction enzyme digests, reaction buffer selected was the most optimal for all enzymes; in which this was not possible, sequential digestions were carried out, with DNA being purified after each reaction with a GFX PCR DNA and gel band

purification kit (GE Healthcare) according to the manufacturer’s instructions.

2.2.2.4 De-phosphorylation of vector DNA

Removal of 5’ phosphate groups from DNA was achieved by adding 1 unit of shrimp alkaline phosphatise (SAP) (Fermentas) to per µg of DNA treated according to the manufacturer’s instructions. SAP was inactivated by incubating the reaction mixture at 65°C for 15 minutes.

2.2.2.5 Conversion of 5’ overhangs restriction enzyme ends to blunt ends

5’ overhangs generated in a restriction enzyme digestion were filled in using large fragment of DNA polymerase I (1unit/µg DNA treated) (Invitrogen) in the supplied buffer together with 0.5mM of each dNTP at room temperature for 15 minutes. The fill-in reaction was subsequently terminated by phenol extraction.

2.2.2.6 Phenol/Chloroform extraction

An equal volume of Phenol/Chloroform/iso-amyl alcohol solution was added to the DNA sample followed by vortexing for 1 minute, the mixture was centrifuged at 16,000 x g in a micro-centrifuge for 2 minutes. The upper layer was then transferred to a clean tube and an equal volume of chloroform/iso-amyl alcohol was added. The mixture was vortexed and centrifuged again as before. The upper layer was

transferred to a clean tube containing distilled H2O making the final volume of 250µl

and stored at -20°C until needed.

2.2.2.7 Ethanol precipitation of DNA

DNA in aqueous solution was precipitated by adding 1/10 volume of 3M sodium acetate pH 5.2 and 2.5 X volume of 100% ice-cold ethanol. After incubation at -70°C for 1 hour, precipitated DNA was collected by centrifuging at 13,200rpm at 4°C for 10 minutes using a tabletop microcentrifuge. Pellets were washed in 80% ice-cold ethanol followed by drying in a vacuum dessicator for 20 minutes. Pellets were then resuspended in 50µl of sterile distilled water.

2.2.2.8 Agarose gel electrophoresis of DNA

DNA molecules were typically separated by elecreophoresis on 1% (w/v) agarose in 1X TBE buffer (89mM Tris base, 89mM Boric acid and 2mM EDTA) containing 0.5µg/ml ethidium bromide. DNA samples were loaded onto the gel in 1X loading buffer (5% (w/v) glycerol, 0.04% (w/v) bromophenol blue and 0.04% (w/v) xylene cyanol). Electrophoresis was carried out in 1X TBE buffer at 70V for 1 hour. DNA fragments were visualised under UV light and imaged using a BioRad Gel/Chemi Doc system with associated software.

2.2.2.9 Agarose Gel Extraction of DNA

DNA bands of interest were excised from the agarose gel using a clean scalpel under UV light. The DNA was recovered using a Qiagen Gel Extraction Kit or a GFX PCR DNA and gel band extraction kit under the instructions recommended by the

manufacturers.

2.2.2.10 DNA Ligation

Ligation of DNA molecules was performed in a final reaction volume of 20-50µl. A 3:1 molar ratio of insert DNA to vector DNA was typically used. This ratio was changed to 5:1 in some particular reactions to increase ligation efficiency. T4 DNA ligase was used according to the manufacturer’s instructions with supplied buffers under recommended conditions.

2.2.2.11 DNA Sequencing

All DNA sequencing was done in-house by the Molecular Biology Service at University of Warwick using an automated ABI PRISM 3130xl Genetic Analyse. Sequencing data were viewed using Chromas Lite 2.0 and analysed using Clone Manager, SciEd Central, v7.04 software.

2.2.2.12 Transfection of plasmid DNA

Lipofectamine 2000 (Invitrogen) was used for liposome-mediated transfection of plasmid DNA throughout the entire study. A ratio of 2µl of reagent per µg of plasmid DNA transfected was used, and this ratio was applied to all transfections in all cell lines used during this study.

All transfections were performed in 12 or 24-well plates. Cells were seeded about 24 hours before the procedure to allow approximately 60-70% of cell confluence by the time of transfection. Transfection reagent and DNA mixture was prepared in pre- warmed OptiMEM medium to make a total volume of 100µl (24-well plate) or 200µl (12-well plate) per well. After incubation for 20 minutes at room temperature the complex mixtures were gently pipetted onto the cells containing serum medium. Cells were then incubated at 37°C in a 5% CO2 incubator for 24 or 40 hours