Entrevista a una profesional encargada del ocio.

Human, plasmid, phage and cosmid DNA were digested with the appropriate restriction enzyme. Digests of human DNA were carried out in a total volume of 40|il. 3-5pg of human DNA was added to 4|il of the appropriate restriction enzyme buffer and 10-20 units of restriction enzyme. The volume was made up to 40pl with distilled water. The restriction endonuclease digests were incubated at 37°C for either 4-6 hours or overnight.

Cosmid, plasmid and phage DNA for electrophoresis were digested in a total volume of 20pl. DNA was added to 2 |liI of the appropriate restriction enzyme

buffer and 10-20 units of enzyme. 2.4.7 Electrophoresis

2.4.7.1 Separation of restriction enzyme fragments

An agarose gel, the percentage of which was determined by the size of the fragments to be separated, was prepared in 1XTAE, brought to boiling point in a microwave oven to dissolve the agarose and allowed to cool to 60°C prior to pouring. Ethidium bromide (final concentration 0.15 mg/ml) was added to the cooled agarose to enable the visualisation of the DNA. The ends of a 20X25 cm BRL gel tray were sealed with tape. A 20 well comb was inserted Into the agarose and the gel allowed to set at room temperature for 1-2 hours.

A volume of lOpI of loading dye was added to each 40pl digest and the total volume loaded into a well. Lambda DNA (Ipg) digested with either BstE II or Hind III was used as a molecular marker. Gels were electrophoresed at 50 Volts for 16-24 hours and photographed on an ultraviolet (UV) light transilluminator against a fluorescent ruler, to mark the relative positions of the bands.

Smaller volumes of restriction enzyme digests were separated on a 11 cm x 13.5 cm NBL gel tray and electrophoresed at 100 Volts for 2 hours.

The vector was digested with the appropriate restriction enzymes and electrophoresed as described in section 2.4.7.1. A UV transilluminator was used to visualise the appropriate fragment, which was cut from the gel in a slice with a scalpel. The gel slice was transferred to a 0.5ml tube which had been plugged with glass wool and with a hole punctured in its base. This tube was then placed inside a 1.5ml appended tube and the DNA eluted by centrifugation at 13, OOOg in a bench microfuge for 10 minutes. The concentration of the eluted DNA was determined spectrophotometrically.

2 4.7.3 Preparation of single copy probe from cosmid and plasmid vectors

Single copy fragments were obtained by digesting the vector with the appropriate restriction enzyme and separating the digested fragments by agarose gel electrophoresis as described in section 2.4.7.I. The gel was photographed prior to Southern blotting (section 2.4.8) and the blotted DNA was then hybridised with radio-labelled total human DNA. The bands which are observed on the X-ray film (X-0MAT-AR5, Kodak) contain repeat sequences, therefore bands which are not visible on the film but are visible on the original photograph should be free of repeat sequences.

2.4.7 4 Separation of PCR products In agarose gels

A 2% agarose solution in 1XTBE buffer containing ethidium bromide (final concentration 0.15 mg/ml) was prepared and poured into a sealed 11 cm x 13.5 cm NBL gel tray. After setting, the gel was placed in an electrophoresis tank, covered with 1XTBE buffer and 5pl of PGR amplified DNA sample

loaded. The gel was electrophoresed at 10 volts/cm for 30-60 minutes. Band sizes were determined by comparison with a 100 bp ladder (BRL) which was also loaded onto the gel at the same time as the PCR product.

2.4.7 5 Separation of microsatelllte polymorphisms by polyacrylamide gel electrophoresis

Denaturing polyacrylamide gels were used for the analysis of PGR amplified CA(n) repeats. A large (42x33cm) BRL glass plate and a small (39x33cm) BRL glass plate were washed in detergent, rinsed and dried. The plates were wiped with 70% ethanol and the smaller of the two plates was coated with a

siliconising fluid (Sigmacote, Sigma). This prevents the gel from adhering to the small plate and ensures that it remains bound to the large plate when the two plates are separated. The gel mould was formed by placing the large plate on a flat surface, arranging two 0.4mm spacers (0.4mm) down the edges and placing the small plate with the siliconised surface facing inwards, on top. The two plates and spacers were carefully aligned and clipped in place with bulldog binder clips.

A denaturing 6% (v/v) polyacrylamide gel (section 2.3.5) was caste. To 90pl of gel mix, 540pl of 10% ammonium persulphate and 60|xl of TEMED were added to catalyse cross linking in the liquid gel. The gel mix was gently poured between the two plates which were placed horizontally, using a 50ml syringe. Two 25 well sharkstooth combs were inserted into the gel, with the flat edge approximately 1cm into the gel and clipped in place. The gel was left to polymerise for 2 hours.

The gel was then placed in a vertical BRL tank and clamped into place. The upper and lower reservoirs were filled with 1XTBE (500mls in each). The sharkstooth combs were removed from the gel and the wells were washed thoroughly with 1XTBE to remove any traces of urea prior to inserting the sharkstooth combs. The sharkstooth combs were inverted so that the teeth just penetrated the gel to form wells. The wells were rinsed again. The sequencing apparatus was connected to a Consort E734 power pack and the gel pre-run at 60W for 30 minutes. The wells were washed out with 1XTBE prior to loading. An 8pl aliquot of each PCR product was added to 2pl of formamide dye and the samples were denatured at 94°C for 5 minutes and then placed on ice. 4pl of each sample was loaded onto the gel and run at 60W for 2-3 hours, depending on the size of the PCR product.

After electrophoresis was complete, the gel assembly was removed from the tank and the smaller plate was levered away. The polyacrylamide gel was blotted onto a piece of 3MM Whatman paper, covered with clingfilm and dried on an ATT A Gel Drying Processor AE-3700. The dried gel was exposed to X-ray film overnight at room temperature.