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DNA molecules can be separated by size by placing DNA samples in a solid matrix, usually agarose or polyacrylamide, and inducing the molecules to migrate through the gel either under a single, static electric field or multiple, variable electric fields. In conventional gel electrophoresis, DNA molecules up to 20kb in size can be separated on standard agarose gels, from 0.6% to 2% concentration, in a single, static electric field. Larger DNA fragments can be separated by using a pulsed field system in which two alternating electric fields, orientated opposite each other, are used. This is called field inversion gel electrophoresis (FIGE) and it enables DNA fragments between 50 and 600kb to be resolved. DNA fragments differing in length by a single

base pair can be resolved on denaturing polyacrylamide sequencing gels. These high- resolution gels fractionate single-stranded DNA fragments on the basis of their size. 2.4.1 Gel Electrophoresis For Southern Analysis

Completely digested genomic DNA was usually separated on 0.8% agarose maxi­ gels (20cm X 25cm) made up in 0.5X TBE with ethidium bromide added to a final

concentration of 0.2|ig/ml. Each digest had 1/lOth volume of loading buffer added to it before loading onto the gel; a DNA size marker was also loaded (Ikb ladder or X Hindi IF). Samples were run into the gel at lOOV for 10 minutes and then at 40 V overnight in 0.5X TBE. Gels were visualised under UV light and photographed. 2.4.2 Field Inversion Gel Electrophoresis

Digested genomic DNA embedded in agarose was run on 1 % ultra-pure agarose gels made up in 0.5X TBE with no ethidium bromide. Blocks were loaded into the wells and sealed using 1% LMP agarose; in addition, 2 pulsed field gel (PEG) ladders were loaded covering the size ranges 0.1 - 200kb and 50 - lOOOkb. Gels were run in 0.5X TBE, which was pre-chilled through a chilling system. The following run conditions were used:

Pre-run: 15 minutes Active run: 44 hours

Forward switch time: 0.3 seconds Reverse switch time: 0.1 seconds Voltage: 190 V

At the end of the run, gels were stained in a 0.5|ig/ml solution of ethidium bromide in deionised water for 15 mins and then destained in deionised water for 30 mins. Gels were visualised and photographed under UV light.

2.4.3 Denaturing Polyacrylamide Gel Electrophoresis

Sequencing reactions (2.9) were run on the Bio-Rad Baserunner system. Before pouring the gel, plates were thoroughly cleaned, initially with detergent, then 70% ethanol and finally rinsed with deionised water, after which they were dried using paper towels. The mirrored plate was treated with Dimethyldichlorosilane solution, to prevent the gel sticking, rinsed with deionised water and dried. The plates were assembled ready to pour the gel. The mirrored plate had 2 clean spacers attached to

the long edges of the plate using double-sided tape, at the top and bottom and the glass plate was placed on top.

The standard mix for a 70ml gel was: 29g Urea

7ml lOX TBE

10ml 40% Acrylamide (38% acrylamide: 2% bis acrylamide)

This was made up to 70ml final volume with deionised water. Incubating the mix at 65°C assists with the dissolving of the urea, once this had dissolved the mix was put on ice. To the cooled gel mix, 420|il of 10% ammonium persulphate and 70|Ltl of TEMED were added, mixed gently and quickly drawn up into a 50ml syringe. The gel was poured, avoiding the incorporation of air bubbles. The comb was put in the top of the gel and the plates clamped together at the bottom, top and over the comb. The gel was left to set at RT for approximately 20 mins and either run straight away or stored at 4°C overnight.

Gels were pre-run in IX TBE at 45W for approximately 15 mins before loading the samples. The comb was removed and replaced, but with the sharks teeth just

entering the gel at the top. Samples were heated at 80°C for 2 mins and immediately loaded onto the gel in the order. A, C, G, T. Gels were run at 45W for 2.5 or 6 hrs. On completion of the run, the glass plate and gel were immersed in fixative (10% acetic acid, 10% methanol in distilled water) for 20 mins. The gel was transferred to 3MM paper and covered with clingfilm before being dried under vacuum at 90°C for 30 mins. The dried gel was then exposed to X-ray film at RT overnight and the film developed.

2.4.4 Separation Of PCR Products On High Resolution Agarose Gels

Using high resolution agarose, which have finer sieving properties, DNA molecules below Ikb in size can be separated with much greater resolution. PCR reactions (2.10) were separated on either 2-4 .5 % Nusieve/Seakem agarose gels or more recently 1.8 - 5% Metaphor agarose gels, the latter producing higher resolution of the amplified products. Typically, the samples were run on 20 x 12.5cm gels made up in 0.5X TBE. All the PCR reaction (20|ll1) was loaded onto the gel, with 2\il of loading buffer, and run at 80V for 5-6 hrs. Gels were stained in a 0.5|ig/ml solution of ethidium bromide in deionized water for 15 mins and destained in deionized water

for 15-30 mins to reduce the background levels of ethidium bromide staining. Gels were visualised under UV light and photographed.