Southern Analysis

Digested DNA was separated, by size, on agarose gels (2.4.1) and transferred to nylon membrane, to which it irreversibly binds. This allows digested DNA to be hybridised with DNA probes many times.

2.5.1 Southern Blotting

After visualisation and documentation, gels were agitated in 0.25M hydrochloric acid (HCl) for 15 mins and rinsed in deionised water. They were then transferred to 0.4M sodium hydroxide (NaOH) for 30 mins, whilst a “ dry” blot was set up. A “ dry” blot consists of 6 pieces of 3MM paper, slightly bigger than the gel, soaked in 0.4M NaOH forming the base of the blot. Denatured gels were placed onto the 3MM paper, making sure that no air bubbles were trapped between gel and paper. The membrane (cut to the same size as the gel) was placed on the gel, again all air bubbles were removed. When blotting using Hybond N+, the membrane was placed directly onto the gel; when using Genescreen Plus, the membrane was soaked in deionised water for 1 min and then transferred to 0.4M NaOH for 15 mins before use. Two more pieces of 3MM paper, soaked in 0.4M NaOH, were placed on top of the membrane followed by a stack of paper towels. A glass plate and weight were then placed on top. The edges of the exposed 3MM paper were sealed with Nesco film to prevent evaporation. Southern blots were left overnight to allow the DNA to transfer to the membrane. On completion of transfer, membranes were rinsed in 2X SSC to remove any agarose, blotted and air-dried. Membranes were stored at 4°C in Saran wrap.

2.5.2 Probe Isolation

Fragments of DNA used as probes were separated from any contaminating

sequences by isolation on agarose gels of a suitable percentage. Ethidium bromide stained DNA fragments were cut out of gels and stored at -20°C for at least 1 hr. A gel slice was incubated at 65°C for 5 mins and transferred to a 0.5ml tube, the bottom of which had been pierced with a needle and plugged with sterile glass wool. The capped 0.5ml tube was then placed inside a 1.5ml tube and centrifuged at 13000 rpm for 5 mins. The eluant in the 1.5ml tube was transferred to a clean tube and an

equal volume of P:C:I was added, mixed and centrifuged at 13000 rpm for 5 mins. The upper layer was transferred to a clean tube and an equal volume of

chloroformiisoamyl alcohol was added, mixed and centrifuged at 13000 rpm for 5 mins. The upper layer was transferred to a clean tube and 0.1 volumes of 3M sodium acetate pH 5.2 and 2 volumes of 100% ethanol was added, mixed and centrifuged at 13000 rpm for 5 mins. The DNA pellet was washed in 70% ethanol, air dried, resuspended in TE pH 8 (usually - 20p,l) at 4°C overnight and the concentration estimated by running an aliquot on a gel.

2.5.3 Radioactive Labelling Of DNA Probes

All radioactive labellings were performed using the Stratagene Prime-It II random primer labelling kit. A standard reaction mix was:

25ng (l-23ql) DNA template 0-23ql Deionised water

lOfil Random oligonucleotide primers

This mix was heated at 100°C for 5 mins to denature the DNA and centrifuged briefly before adding:

lOql 5X Primer buffer (dCTP) 5^1 [a “ ‘’ ] dCTP

Ifil Exo (-) Klenow (5U/p.l)

This was mixed gently with a pipette tip and incubated at 37°C for 10 mins. To end the reaction, 2|li1 of Stop solution was added. The labelled probe was cleaned, to remove any unincorporated radioactivity and nucleotides, using Pharmacia Nick columns (prepacked, disposable Sephadex G-50 DNA grade columns). The cap was removed from the top of the column and the liquid poured off before being rinsed with equilibration buffer. The bottom cap was removed and the column supported over a suitable receptacle. The column was filled with equilibration buffer and allowed to drip through. The labelled sample was loaded onto the gel bed (up to lOOql volume) and 400|il of buffer solution was loaded on top and allowed to enter the gel bed. A tube was placed under the column for sample collection and a further 400|il of buffer solution added to the column. The labelled eluant (400p.l) was now ready to be used in the hybridisation procedure.

2.5.4 Southern Hybridisation

Southern hybridisations were performed using Genescreen Plus and Hybond-N+ membranes, different hybridisation buffers were used for each membrane.

Membranes were pre-wetted with 2X SSC and blotted briefly. For each 20cm x 20cm membrane, 10ml of hybridisation buffer was used. Membranes were prehybridised in hybridisation buffer at 65°C for at least 1 hr. The clean, labelled probe was incubated at 100°C for 5 mins, to denature, (when using Genescreen Plus membranes, salmon sperm DNA was added to the probe so the final concentration in the hybridisation buffer was O.lmg/ml) and then placed on ice. The denatured probe was mixed with 5ml of pre-warmed hybridisation buffer, this was added to the prehybridising membranes and incubated at 65°C overnight with gentle agitation. 2.5.5 Membrane Washing

Hybridising membranes were removed from the hybridisation buffer and placed into a wash solution of 2X SSC; 0.1% SDS, and briefly washed at RT. They were then transferred to fresh 2X SSC; 0.1% SDS and incubated at 65°C for 20 mins with gentle agitation. Membranes were monitored using a Geiger-Müller tube (GM-tube), and washed at increasingly higher stringencies until a reading of ~ 5-10 counts per min (cpm) was achieved, i.e. Just above the background level. The next stringency wash would be IX SSC; 0.1 % SDS, followed by either 0.5X or O.IX SSC; 0.1 % SDS. Membranes were covered in Saran wrap and exposed to X-ray film at -70°C overnight using cassettes with intensifying screens. X-ray fihns were developed and exposed for a longer period if bands or signal were faint. Membranes were washed at a higher stringency if the developed X-ray films revealed non-specific binding of the probe.

2.5.6 Membrane Stripping

Membranes were usually hybridised with a number of DNA probes and stripped of DNA probe before being rehybridised.

To strip HybondN-t- membranes, a boiling 0.5% (w/v) SDS solution was poured onto the membranes and allowed to cool to RT.

To strip Genescreen Plus membranes, they were incubated in 0.4M NaOH at 42°C for 30 mins followed by 30 mins incubation in O.IX SSC; 0.1% SDS; 0.2M Tris-HCl pH 7.5 at 42°C.

Stripped membranes were exposed to X-ray film at -70°C overnight to check that DNA probes had been removed.