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Typically lOwg of vector DNA was linearised with the appropriate restriction enzyme(s) and the efficiency of the enzyme(s) was tested on agarose/ethidium bromide gels. Linear vector was phenol/chloroform extracted and ethanol precipitated before resuspending in 90m1 of lOmM Tris pH 8.3, IOmI of 10 x phosphatase buffer (Boehringer

Mannheim) and 1 unit of calf intestinal alkaline phosphatase and incubating at 37°C for 30 minutes. The reaction was stopped by addition of lOOwl of 2 x stop solution (1% SDS, lOmM EDTA and 200wg/ml proteinase-K) and incubation at 56°C for 30 minutes. Vector was then phenol/chloroform extracted and ethanol precipitated.

ii). DNA Fragm ent Purification

DNA fragments were purified using the Geneclean II kit (BIO 101 Inc). Purification was carried out according to the protocol obtained with the kit

Briefly the appropriate fragments were cut from TAE gels in minimal volumes of agarose and dissolved in 3 volumes of sodium iodide at 50°C for 5 minutes. 5m1 of glassmilk was added and the mixture left on ice for 5 minutes before pelleting the matrix at 12,(XX)g for 10 seconds. The glassmilk matrix was resuspended in 5(X)m1 of -20°C New wash and respun and resuspended 3 times. After removing all the wash solution the pellet was resuspended in lOwl of dH20 and incubated at 50°C for 3 minutes, then spun at 12,(XX)g for 30 seconds. DNA in the supematent was retained for ligation.

iii). Ligations (All solutions were sterile).

Approximately 200ng of vector DNA and 5 x the molar concentration of insert DNA were ligated in I O m I volumes containing 1 x ligation buffer (Boehringer Mannheim)

and 1 unit of T4 DNA ligase at 14°C overnight Molecular weight marker DNA (X Hind-in digest) was also ligated and run on agarose/ethidium bromide gels to test the efficiency of the ligase.

iv). Recombinant Selection (all solutions were sterile).

Ligated DNA was used to transfomi competent cells (see 2.2.2.) and white colonies were picked and grown for 2 hours at 37°C in 200u\ of LB containing 50Mg/ml ampicillin (Berk) in 96 well plates. 0.5m1of each clone was then spotted onto Hybond N. To the

remainder of each clone 2 drops of glycerol was added and these were frozen at -70®C. Ing of Bluescript DNA was also used to transform competent cells in order to test their DNA uptake and colour selection efficiency.

Nylon filters were prepared for hybridisation with 5 minute washes, twice in 0.5M sodium hydroxide, twice in IM Tris pH 7.5, twice in 1.5M sodium chloride and 0.5M Tris pH 7.5 and finally for 5 minutes in 2 x SSC. The DNA was then cross linked to the filters by baking for 2 hours at SO^C. 1m1 of DNA insert was oligo labelled (see 2.2.5.) and used to probe filters, which were prehybridised in 6 x SSC, 1 x Denhardts and lOOMg/ml denatured herring sperm DNA at 65°C for 30 minutes. Hybridisation was carried out in the

same solution plus denatured probe at 65°C overnight. Filters were washed twice in 1 x SSC and 0.5% SDS at 65°C for 30 minutes prior to autoradiography for 1 hour. Positive clones were grown up fiom glycerol stocks for miniprep and restriction enzyme analysis.

2.2.7. Transfections (All solution were filter sterilized).

8 X 10^ cells (1 X 10^ cells in conditioned medium for stable cell lines) on a 90mm plate were transfected by the calcium phosphate procedure of Gorman (Gorman, 1985). Briefly cells were transferred to DMEM supplemented with 10% v/v foetal calf serum 2 hours before transfection. Plasmids and herring sperm carrier DNA were sterilized by ethanol precipitation and 70% v/v ethanol wash and typically lOug of each was added to each 90mm plate. 4 hours after transfection cells were washed twice in PBS and the standard growth medium was replaced. Cells were harvested by scraping from the plates 48 hours after transfection for the preparation of RNA or for CAT assays.

3T3 cells were transfected with the following modifications to increase the efficiency of DNA uptake. Calcium/phosphate/DNA precipitates were added to plates from which the media had been removed and left at 37°C for 15 minutes before replacing the medium. After 4 hours the medium was removed and replaced with 2.5ml of HBS and

15% v/v glycerol for 2 minutes at 37°C. The cells were then washed as above.

2.2.8. Southern Blotting

Agarose/ethidium bromide gels were prepared for Southem blotting by washing for 15 minutes in 0.5M sodium hydroxide and 1.5M sodium chloride, 15 minutes in 3M sodium chloride and 0.5M Tris pH 7 and 5 minutes in 2 x SSC. Blotting onto nylon filters was carried out overnight using 20 x SSC as blotting buffer. DNA was cross linked to blots by UV irradiation on a UVP transilluminator for 5 minutes. Filters were prehybridised in 6 x SSC, 10% w/v dextran sulphate (Pharmacia), 0.1% w/v SDS, 5 x Denhardts and lOOwg/ml denatured herring sperm DNA (Sigma), for 2 hours at 65°C in a Hybaid MAXI hybridisation oven. Hybridisation to denatured, oligo labelled probe (2.2.5.) was carried out in the same solution overnight at 65°C.

30 minute washes were carried out typically at 65°C in 1 x SSC and 0.2% w/v SDS followed by decreasing concentrations of SSC to 0.1 x or until sufficient washing was achieved for autoradiography.

2.2.9. DNA Sequencing

DNA sequencing was performed using double stranded DNA templates with the 'Sequenase' (Tabor & Richardson, 1987) version 2.0 kit (United States Biochemical) and according to the instructions supplied with the kit. DNA was obtained by large scale plasmid preparation (2.2.3.Ü.).

Sequencing products were seperated by electrophoresis through 6% polyacrylamide/urea gels prepared using sequagel solutions (National Diagnostics). These gels were typically run at 30W in 1 x TBE buffer.