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cDNA and whole cosmid inserts were completely sequenced by the shotgun method of Bankier (Bankier et al., 1987). The DNA was isolated, self ligated, sonicated, end repaired and ligated into Smal-cut M13mpl8.

2.11.1. Vector preparation

5|Xg M 13m pl8 replicative form was digested with 20U Smal, using the appropriate digestion buffer in a total of 50pl for 1 hour at room temperature. A 0.5|il aliquot of the reaction was run on a 0.8% agarose gel to assay for complete digestion. After complete digestion Ipl 0.5M EDTA pH 8.0 was added to the reaction. The solution was extracted with phenol and the DNA precipitated by addition of 5|il 3M NaAc pH 5.2 and 2.5 volumes of ice cold ethanol for 10 mins at -70°C. The tubes were centrifuged for 10 mins in a microfuge, the supernatant discarded and the pellet washed in 70% ethanol. The centrifugation was repeated, the pellet dried under vacuum and resuspended in 25|il TE pH 8.0. 20U CLAP was added and the reaction incubated for 1 hour at 37°C.

The vector was purified by electrophoresis on a 0.8% agarose gel in IX TAE containing 0.5|Xg/ml ethidium bromide. The gel was run at 50V for 2 hrs and visualised under uv light. The linear vector band was excised from the gel using a scalpel and purified by electroelution. The agarose block was transferred to a dialysis bag containing a minimal amount of IX TAE. The bag was sealed at both ends and placed in an electrophoresis tank containing IX TAE. Electrophoresis was carried out at lOOV for 3-4 hrs by which time the DNA had passed out of the

gel into the surrounding solution. The solution was collected, phenol, PCIA, CIA extracted, ethanol precipitated and resuspended in TE pH 8.0 at 20ng/pl.

2.11.2. Insert preparation

~50|ig of either the cDNA or cosmid clone of interest were digested with 10 fold excess o f the appropriate restriction enzyme under optimal conditions to obtain complete digestion. The digested products were run out on a 0.8% agarose gel in IX TAE until separation of the desired bands had been achieved. Either the cDNA insert or the cosm id fragm ent was excised from the gel using a scalpel. Purification was achieved by electroelution and phenol extractions as described in section 2.11.1. The resulting, pure fragments were resuspended in 70pl TE pH 8.0 and 0.5|il run on an analytical 0.8% agarose gel to estim ate the DNA concentration.

10|ig of the fragment was self-ligated in lOOpJ total volume by 1 X 10^ U of T4 DNA ligase in IX ligation buffer (NEB) at 14°C for 16 hrs. The following day 5li1 of the ligation reaction was run out on a 0.8% agarose gel, 8V, overnight. To

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ensure efficient self-ligation A H indlll^as a m arkej. If the self-ligation was effective the fragments were diluted to a total volume of 3(X)}il with low TE pH 8.0 and sonicated to give fragment sizes of -5 0 0 bp ( 3 X 7 second bursts, setting 6, 1 min cooling between bursts, on ice). lOpl of the sonicated DNA was run on a 1.5% agarose gel using 100 Base-Pair Ladder (Pharmacia) to check that fragment sizes of the desired length had been achieved. If not, sonication was repeated, checked, and the DNA ethanol precipitated overnight at -20°C. The sonicated fragments were then washed with 70% ethanol, vacuum dried and resuspended in 60|il low TE pH 8.0. The fragm ent ends were repaired with 16 and 20 units (5U /|il) of Klenow and T4 DNA polym erase respectively, and I p l each of lOOmM dATP, dCTP, dGTP and dTTP added. The DNA was size fractionated in a 0.8% agarose gel, excising fragments in the size range 350-700 bp. These were purified by phenol extraction. The DNA was ethanol precipitated and resuspended in 50pJ low TE pH 8.0.

2.11.3. Ligation

Four ligation reactions were set up for each insert containing 0.1, 1 ,2 and 4fil of insert DNA and 20ng vector DNA. Ligations were carried out in a total o f 20pl

using 2 X 10^ U T4 DNA ligase in the appropriate buffer conditions at 14°C overnight. Control ligations were also set up, one containing no insert DNA and the other containing no insert DNA, and no ligase. Ligations were transfected into JM lOl competent bacteria.

2.11.4. Preparation of competant cells

A single colony of JMlOl bacteria was inoculated into 5ml of 2YT medium and grown overnight, shaking, at 37°C. 300pl of this overnight culture was added to 30ml 2YT medium in a 500ml flask and grown to an OD^oo of 0.4-0.6. The culture was transferred to a Falcon 50ml disposable tube and centrifuged at 2000rpm for 10 mins at 4°C in a Heraeus minifuge T. The pellet was resuspended gently in 2.5ml cold TFB and incubated on ice for 15 mins. lOOpl DMSO was added to the suspension and incubated on ice for 15 mins. lOOpl of DTT/KAc was added and, after 10 mins, a further lOOjxl of DMSO. The cells were kept on ice until needed for transformation.

TFB: lOmM methyl ethane sulphonate

lOOmM rubidium chloride 45mM manganese chloride

lOmM calcium chloride

3mM hexaminecobaltic chloride

DTT/KAc: 2.25M dithiothritol

40mM potassium acetate pH 6.0

2.11.5. Transformation

400|il competant cells and 20pl ligation reaction were added to a sterile glass culture tube and incubated on ice for 45 mins. The cells were heat shocked at 42°C for 3 mins and 6ml molten top agar (45°C), lOOjxl 2% Xgal and 50pl 2.5% IPTG added. The bacteria in top agar were quickly poured onto a pre-warmed (37°C) agar plate and swirled to effect an even distribution. The top agar was allowed to set and the plates incubated at 37°C overnight.

Top agar: lOg bactotryptone 8g sodium chloride 8g bacto agar

to 1 litre with distilled water

X-gal: 2% 5-bromo-4-chloro-3-indolyl-P-galactoside in DMF

(dimethylformamide)

IPTG: 2.5% isopropyl-P-D-thiogalactopyranoside in water

2.11.6. Template preparation

A single colony of JM lOl was inoculated into 10ml 2YT and grown, with shaking, at 37®C overnight. This was diluted 1:100 with 2YT. Individual plaques were picked into 1.5ml aliquots of diluted JMlOl and grown at 37°C for 4.5-5.5 hrs with vigorous shaking. The bacterial cultures were pelleted by centrifugation in a microfuge for 5 mins and the phage-containing supernatant transferred to a fresh tube. The phage were precipitated by the addition of 200|il 20% PEG solution. The supematant/PEG was centrifuged for 5 mins in a microfuge and the PEG removed. The tube was respun for 30 seconds and all residual PEG removed. The phage pellet was resuspended in lOOjil TE pH 8.0 and extracted with an equal volume of phenol to remove the phage coats. The aqueous layer was removed and the DNA ethanol precipitated. The DNA was resuspended in 30pl TE pH 8.0 and 5|i.l used directly for sequencing as described in section 2.10.

20% PEG: 20g polyethylene glycol (8000mw)

14.6g sodium chloride

distilled water to volume of 100ml