Electrophoresis

a Agarose gel electrophoresis

Agarose gels were made with 1-2% agarose in IxTAE containing l/xg/ml EtBr. The solution was boiled and allowed to cool before being poured into a gel former with a comb to form wells for loading DNA samples. The gel was left to set, the comb was removed and the gel placed in a horizontal electrophoresis tank containing IxTAE. The samples to be loaded were first mixed with one tenth volume lOx loading buffer. After sample loading, the gel was run at a suitable voltage to separate the DNA fragments and the resulting bands were then visualised using U.V. transillumination.

lOx TAE buffer: 0.4M Tris O.OIM EDTA

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pH 8.0 with glacial acetic acid

lOx loading buffer: 0.25% bromophenol blue (w/v) 25% Ficoll 400 (w/v)

O.IM EDTA

b Denaturing polyacrylamide gel electrophoresis for DNA sequencing

0.4mm spacers were placed between two clean plates, one of which had been siliconised using Repelcote (BDH) and these were then taped together to prevent leakage of the acrylamide solution. 75mls of 6% acrylamide solution was prepared with 19:1

acrylamide:bisacrylamide solution, 42% urea and IxTBE. The solution was warmed at 37°C to dissolve the urea. Polymerization was achieved by the addition of 75/xl of a crosslinking agent, TEMED (NN N ’N ’-tetramethylethylenediamine) and 75^1 of freshly made 25% ammonium persulphate solution. The solution was then poured carefully between the glass plates, avoiding the formation of bubbles. Two 0.4mm sharks tooth combs were inserted upside down and clipped in place to form a horizontal surface at the top of the gel. Once the gel had set (approx. 1 hour) it was untaped, placed in a vertical electrophoresis apparatus and the combs were replaced with the teeth forming small wells at the surface of the gel. The gel was pre-run in IxTBE until warm (approx. 2 0 minutes) and the wells were flushed out to remove any urea which may

have leached out of the gel. Samples to be loaded were mixed with a 0.5 volume of formamide dye, boiled for 3 minutes, immediately placed on ice and quickly loaded. The gel was run at a constant power of 60W (maximum current 55mA, maximum voltage 2000V) until the required separation was achieved. After the appropriate time

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the gel was transferred to 3MM filter paper (Whatmann) and dried under vacuum at 80°C for at least 1 hour. Since the radioisotope used in sequencing was autoradiography was then carried out overnight at room temperature.

lOx TBE: 89mM Tris-borate

2mM EDTA pH 8.3

Formamide dye: 100ml deionized formamide

0.1% xylene cyanol 0.1% bromophenol blue

20mM EDTA

c Non-denaturing polyacrylamide gel electrophoresis for bandshift assays

Seventy-five millilitres of acrylamide solution was prepared containing 7% 30:1 acrylamide:bisacrylamide and 0.5x TBE. This was polymerized with 75^1 TEMED and 75/d of freshly prepared 25% ammonium persulphate. The gel was poured between two clean glass plates (2 0x2 0cm) separated by 1mm spacers and a wide toothed comb was

inserted and clipped in place. Once the gel had set it was untaped and pre-run for 30 minutes (and for not longer than 1 hour) at 4°C. The samples were then loaded and the gel run for a further 3 to 4 hours at 150V (maximum current 20mA) at 4°C, dried on to DE81 paper (Whatmann) and, since the isotope used was autoradiography was carried out at -70°C overnight.

72 2.2.6 PCR

Standard conditions were used for the polymerase chain reaction (PCR) (Saiki et al 1988; Mullis and Faloona 1987). 200-500ng of genomic DNA were used as template with 200ng of each primer in a 40/d volume. The PCR contained 200/xM each of d i I P, dGTP, dCTP and dATP, lOmM Tris-HCl (pH8.3), 1.5mM MgCl^, 50mM KCl, 10% dimethyl sulphoxide and 0.8 units BRL Taq polymerase. Samples were overlaid with paraffin oil and typically underwent one cycle of 5 minutes at 95°C, 1 minute at 55°C and 2 minutes at 72°C followed by 30 cycles of 1 minute at 95°C, 1 minute at 55°C and 2 minutes at 72®C on a Cambio Intelligent Heating Block. Primers were obtained from The Advanced Biotechnology Centre, Charing Cross and Westminster Medical School or Severn Biotech Ltd, Kidderminster.

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TABLE 1 Polymerase chain reaction conditions and primer sequences

G e n e P C R p u r p o s e P r i m e r s e q u e n c e ( 5 ’ - 3 ’ ) R e a c t i o n C o n d i t i o n s * D e n a t u r i n g A n n e a l i n g E x t e n s i o n C y c l e N o F r a g m e n t S i z e ( b p ) F a c t o r V I I D e t e c t i o n o f A r g / G l n 3 5 3 p o l y m o r p h i s m G G G A G A C T C C C C A A A T A T C A C A C G C A G C C T T G G C T T T C T C T C 9 5 ° C ( I m i n ) 5 5 ° C ( I m i n ) 7 2 ° C ( 2 m i n ) 3 0 3 1 2 j S - f i b r i n o g e n P r o d u c t i o n o f i n s e r t s f o r t r a n s i e n t e x p r e s s i o n c o n s t r u c t s A A A T A A G C T T T G C T G G C C A G C A A A A C T T A T T T A 9 5 ° C ( I m i n ) 5 5 ° C ( I m i n ) 7 2 ° C ( I m i n ) 1 3 0 0 2 4 0 * R e a c t i o n m i x t u r e s w e r e h e a t e d i n i t i a l l y t o 9 5 ° C f o r 5 m i n u t e s t o d e n a t u r e D N A

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