Nucleic acids DNA.

1.1 Genomic DNA preparation.

Tissue samples (~ lOOmg) were carefully homogenised with a glass mortar and eppendorf tube in 430/xl of tail mix buffer. To this, 50/il of 10% SDS, and 20fi\ of proteinase-K (lOmg/ml) were added with gentle mixing. The samples were incubated at 55®C overnight, followed by a Ihr incubation at 37®C with 10/il of lOmg/ml DNAse-free RNAse. The DNA was cleaned by 2x phenol/chloroform extractions, plus one chloroform extraction, and precipitated from the aqueous layer with 0.6 volumes of isopropanol. The DNA was hooked out using a sterile glass pasteur pipette, washed in 70% ethanol, and air-dried for 5 minutes. It was then resuspended in 50-200/^1 of TE pH 7.4, and stored at -20“C until needed.

DNA from tail cuts of mice was obtained as above, with the exception of the homogenisation step, while cells from culture were resuspended in tail mix, before preceding as described.

1.2 DNA fragment purification.

All DNA fragments for either cloning or DNA probes were isolated using the GELase™ (Cambio, Cambridge) or QÏAEX™ (QIAGEN) protocols.

QIAEX™ method: The DNA was run on an ethidium bromide (Ifig/wl) stained, IxTAE/agarose (0.8-1.5%) gel, and the desired band removed with a sterile scalpel blade. The gel slice was weighed in an eppendorf tube, and 300|il of QXl™ solution per lOOmg of gel was added. To this, 10-20/d of QIAEX™ resin was added and the sample was incubated at 50®C for 10 min. After centrifugation at 14K rpm for 30 seconds the supernatant was removed. 2x washes with QX2™ solution and QX3™ solution followed, with centrifugation at 14K rpm after each wash. The QIAEX™ resin pellet was then air-dried, before the DNA was eluted in 20-50/d of TE pH 7.4.

IxTAE/low melting point agarose (0.8-1.5%) gel, and the desired band removed with a sterile scalpel blade. The gel slice was weighed in an eppendorf tube and 1/tl of 50x GELase"^ buffer added per 50mg of gel. The gel slice was then completely melted at 70*C (approx. 20 min). The sample was then equilibrated to 42*C for 10 min., before 1 unit of GELase^ enzyme was added per 300mg of 1% LMP agarose gel. Incubation was from Ihr to overnight. One volume of 5M ammonium acetate, followed by 4x the original volume of room temperature absolute ethanol was then added. The DNA was pelleted by centrifugation for 30 min. at 14k rpm, and the supernatant removed. After air-drying the DNA was resuspended in 10-50/a1 of TE pH7.4.

1.3 DNA quantitation.

For concentrations of DNA thought to be in excess of 300ng//tl, the sample was diluted 1:200 and the absorbance was read at 260/280nm on a spectrophotometer. For samples less than 300ng//tl, 1-2/il of the DNA was run on a 1 % agarose/IxTAE ethidium bromide (l/^g/^l) stained gel, alongside known amounts of standard DNA. The concentration of the sample was then estimated by comparing the intensity of its fluorescence to that of the standards, under a UV light.

1.4 DNA restriction digests.

DNA digestion with restriction endonucleases were performed using the optimal conditions as recommended by the paricular manufacturer. Digests were performed using Ix buffer, DNA (lOng to and 0.5-5 units of enzyme per fig of DNA. The total vetvme of enzyme was always limited to no more than 1 0% of the reaction volume to avoid glycerol

inhibition of digestion. Incubations were at 37"C, unless otherwise instructed, for periods from Ihr to overnight.

1.5 Slot blot analysis of DNA.

Slot blot analysis of DNA from tissue samples. Sfig of DNA was added to dHjO, to a final volume of 180/il. To this 20/d of 4M NaOH was added, mixed and left at room temperature for 5min. The slot blot manifold was prepared by laying 2 sheets of 2x SSC soaked Watman

3MM paper onto the lower half of the apparatus. On this was placed a pre-wet nitrocellulose filter (0.45-micron pore size) that had been soaked in IM ammonium acetate for lOmin. The top manifold was fitted and clamped in place. The vacuum line was attached and turned on. Under suction, each slot was filled with IM ammonium acetate and allowed to empty. To the DNA samples 200/il of 2M ammonium acetate was added and mixed. The 400/d sample was then added to the slot and allowed to pass through onto the filter. Known amounts of plasmid DNA added to 5/ig of normal mouse genomic DNA were used as copy number controls. After the last sample the filter was allowed to dry under suction for 5min. The vacuum line was then removed. The filter was air-dried then baked in an 80°C oven for 2hr. The blots were hybridised in conditions as used for Southern bolt analysis.

1.6 Southern blot analysis of DNA.

Between 5-20/ig of genomic DNA was digested overnight with the appropriate restriction enzyme(s). The sample was then mixed with 0.2 volumes of orange-G loading dye, and loaded onto an ethidium bromide (l/tg//il) stained agarose (0.8%-1.2%)/TAE (Ix) gel. Bacteriophage Lambda DNA, digested with BstEII, was used as DNA size markers, and plasmid DNA of known concentration was added to normal non-transgenic mouse DNA as copy number controls. The samples were run through the gel at between 1-5 V/cm, until the orange G front had reached the end of the gel. The gel was photographed and the position of the Lambda size markers were noted. The gel was inverted and soaked in 0.25M HCl for 20 min at room temperature, with gentle agitation. The gel was then washed twice with 0.5M NaOH/1.5M NaCl for 20min., followed by 2x 20min. washes in 0.5M Tris pH 7.4/1.5M NaCl. The inverted gel was then placed onto a wick comprised of a piece of Watman 3MM paper dipped into a tray of 20x SSC. The following was placed on top of the gel ensuring that no air bubbles were trapped; 1 piece of nitrocellulose or nylon (Nytran) filter soaked initially in HgO then in 20x SSC for 20 min., 2-4 20x SSC-soaked pieces of 3MM paper, 20-40 sheets of dry 3MM paper, a 5cm stack of dry paper towels, a glass plate and two 500ml bottles half filled with water (weight approx. 0.5kg). This blotting set-up was left for 12-16hr. The apparatus was dismantled and the position of the wells were marked on the filter with a water-proof pen. The filter was then baked at 80*G for 2hr. and hybridised as described below.

1,7 Oligo-labelling of DNA probes (random priming).

DNA fragments from 100-5000bp were used as DNA probes. lOOng of DNA in 7/a1 of ddHgO was denatured by boiling at 100XÜ for 5min, then placed on ice. To this, 12/d of 2xOLB mix, 1/d of 1 mg/ml BSA, 3/d of a-dATP and 1/d of Klenow enzyme (5U//tl) were added. The mix was incubated at 37**C for 30min, with the reaction terminated by the addition of 10 /il of 0.25M EDTA. The labelled DNA was separated from unincorporated nucleotides by spinning the DNA through a 2x SSC-equilibrated G50 sephadex column. Probes with a specific activity above 1x10* cpm//ig of DNA were used.

1.8 End-labelling of DNA probes.

DNA probes were end-labelled using T4 polynucleotide kinase. The DNA fragment was prepared by leaving a 5* overhang at the end of the DNA that is to be labelled, by digestion with an appropriate restriction endonuclease. The phosphate was removed from this S’ end by incubation with l/il of calf intestine alkaline phosphotase (lU//d) for 30 min at 37**C. The DNA was cleaned using Ix phenol/chloroform plus Ix chloroform extraction, followed by an ethanol precipitation. The labelling reaction involved 100 ng of this prepared DNA in 10/d. To this 4/d of 5x kinase buffer, 5/d of ydATP, and 1/d of T4 polynucleotide kinase were added, and the mixture incubated at 37**C for 30 min. The reaction was terminated by adding 1/d of 0.5M EDTA and 1/tl of 10% SDS and the labelled DNA separated from the unincorporated nucleotide by spinning it through a G50 sephadex column. Probes with a specific activity greater than 1x10^ cpm//tg were used.

1.9 Hybridisation of DNA filters.

DNA filters were pre-wet in 2x SSC then placed in Hybaid hybridisation bottles. The filters were pre-hybridised for 2hr at 6 8"C in 20ml of DNA pre-hybridisation solution without the

radio-labelled probe. The pre-hybridisation solution was removed. The probe was then added to 20ml of DNA hybridsation solution and incubated overnight at 6 8®C. The filters were then washed

twice for 20 min in 2x SSC/0.1% SDS and twice for 20 min in 0.2x SSC/0.1% SDS. The filter was then covered in Saran wrap and exposed on film (4hr-lwk) or on the Molecular Dynamics

Phosphorimager.

1.10 Polymerase Chain Reaction (PCR).

Great care was taken to avoid DNA or RNA contamination, the reaction usually being set up in a different lab or tissue culture hood. 5^1 of cDNA sample (see above), 200ng of genomic DNA or 0.2pg of plasmid DNA was used per reaction. To this lOQng of sense and antisense primer, and 25/d of 2x PCR mix was added and made up to 50/tl with H^O. The PCR conditions were 5 min at 94®C, then, 40 cycles of 94®C for 30 sec, 52®C for 30 sec, and 72®C for 1 min, with a 72®C incubation for 10 min to end. This was performed by a Techne PHC-2 PCR machine, and the results were visualised on an ethidium bromide staining agarose/TAE gel.

Primers: Nef sense - 5*-GCCTGTACTGGGTCTCTCTGG-3’

Nef antisense - 5’-CCCAGGTTTCCAAGGCATTCG-3’ HPRT sense - 5 -CAGAGGACTAGAACACCTGC-3'