Procesos metalúrgicos

II.8.A. Restriction and dephosphorylation of plasmid vector

The plasmid vector pUC8 (see Appendix I) was used in all cases for the subcloning of DNA fragments excised from the lectin- positive bacteriophage lambda clones. Normally 1 ug of pUC8 DNA was linearised with the appropriate restriction enzyme in the usual way. The digest was then extracted once with an equal volume of TE-equilibrated ltl (v/v) phenol/chloroform mixture. The DNA was precipitated by the addition of a tenth volume of 3 M sodium acetate pH 4.8 and 2 volumes of ethanol. After chilling on dry ice for 20 min, the DNA was pelleted by centrifugation for 10 min in a microcentrifuge, dried iji vacuo and redissolved in 20 ul of sterile distilled water. Removal of the terminal S' phosphate groups of the linearised vector was achieved using the method described by Maniatis £t al (1982). To the resuspended DNA was added 2.5 ul of 10 x phosphatase salts (10 mM MgCl^, 1 mM Z n C ^ ) » 2.5 ul of 0.5 M glycine (made to pH 9.4 with NaOH) and 1 ul of 1 u/ul calf intestinal phosphatase. The mixture was incubated for 15 min at 37°C, and then for 15 min at 56°C. Following the addition of a further 1 ul of 1 u/ul calf intestinal phosphatase, the incubation procedure was

repeated. 20 ul of sterile distilled water was then added to the preparation, followed by 5 ul of 10 x STE (0.1 M tris-HCl pH 8.0, 1 M NaCl, 10 mM EDTA) and 5 ul of 0.1 M nitrilotriacetic acid. The tube was heated at 6 8°C for 15 min and 6 ul of 3 M sodium acetate pH 4.8 was added. The preparation was extracted 3 times with an equal volume of 1:1 (v/v) TE-equilibrated phenol/chloroform mixture and 120 ul of ethanol added to precipitate the DNA. The precipitate was then pelleted in a microcentrifuge, washed 3 times with 70Z (v/v) aqueous ethanol, dried ^n vacuo and redissolved in 10 ul of TE. 1 ul of this preparation was electrophoresed on an agarose gel as described in section II.10.B. in order to verify that complete linearisation had taken place, and to estimate the concentration of DNA in the preparation. Dephosphorylated vector DNA was stored at -20°C.

II.8.B. Gel isolation of DNA fragments

This was carried out using a similar procedure to that described by Maniatis e£ al (1982). Digested DNA was electrophoresed, in this case on a 0.7Z (w/v) low melting point agarose, 1 x TBE (89 mM tris, 89 mM boric acid, 20 mM EDTA) gel. Both the gel and the running buffer (also 1 x TBE) used contained 0.5 ug/ml ethidium bromide, thus eliminating the need for staining after electrophoresis. The digested DNA was visualised on a uv transilluminator

and the desired DNA bands identified by means of size markers also present on the gel. A glass plate was placed under the gel and the relevant DNA bands were excised in a minimum volume of agarose using a razor blade. Each slice of agarose was then placed in an Eppendorf tube and 150 ul of TE added. The agarose was then melted by incubating each tube at 50°C for 10 min and 150 ul of TE-equilibrated phenol, pre-incubated at 50°C, was immediately added to each tube. The tubes were then centrifuged for 2 min in a microcentrifuge to separate the aqueous and organic phases. A further extraction was carried out using 150 ul of TE-equilibrated 1:1 (v/v) phenol¡chloroform mixture. 3 ul of 1 ug/ul E. coll tRNA was added to the final aqueous phase and the nucleic acids precipitated by the addition of a tenth volume of 3 M sodium acetate and 2 volumes of ethanol. Finally, after the tubes had been left on dry ice for 20 min, the precipitate was pelleted by centrifugation for 10 min in a microcentrifuge, washed once with 70Z (v/v) aqueous ethanol, dried i_n vacuo and redissolved in 10 ul of TE. The yield of DNA was assessed in the same way as was described for the dephosphorylated vector. Purified DNA fragment preparations were stored at -20°C.

DNA fragments isolated from bacteriophage lambda clones were ligated into dephosphorylated linearised DNA of the plasmid pUC8 in a 10 ul reaction mixture containing 1 ul of the same 1 0 x buffer as was described previously (section II.5.E.). A total of 100 ng of DNA, consisting of vector and insert DNAs in equimolar amounts, was used in each reaction. Incubation was carried out overnight at 15°C.

II.8.D. Transformation of ligated plasmids into E. coll

A method based on that of Mandel and Higa (1970) was used. 50 ml of L-broth in a conical flask was inoculated with 1 ml of an overnight culture of E.coli strain DH5»< and shaken at 37°C until an Ag0o of 0.48 had been reached. The culture was then placed on ice for 5 min prior to centrifugation at 2 000 g, 4°C for 5 rain. The resultant cell pellets were gently resuspended in a total of 25 ml of ice-cold 10 mM CaCl2 . The tubes were left on ice for 15 min and then centrifuged again at 2 000 g, 4°C for 5 min. Finally the cell pellets were gently resuspended in a total of 2 ml of ice-cold 50 mM CaCl2 and kept on ice for a minimum of 30 min prior to use. Aliquots of 200 ul were used for each transformation. Normally 2

transformations were carried for each ligation reaction; one using 5 ul of the ligation mixture and the other using 0.5 ul of the mixture. Transformations were carried out

in separate Eppendorf tubes which were left on ice for 30 min after the competent cells had been inoculated with DNA. The cells were then heat shocked at 42°C for 2 min and 1 ml of L-broth was added to each tube. Following incubation for 1 h at 37°Ct the cells were pelleted by centrifugation for a few seconds in a microcentrifuge. Each pellet was then gently resuspended in 100 ul of L- broth and the cell suspension plated out onto 1.5Z (w/v) L-agar plates containing 50 ug/ml ampicillin. In some cases the agar was supplemented with 0.2 mg/ml IPTG and 0.17 mg/ml X-gal. Under such conditions non-recombinant transformants give rise to blue colonies, whereas colonies from recombinant transformants are white. Thus any non­ recombinant clones could be discarded at this stage.