Lectura de resultados

DNA was recovered from SeaPlaque agarose gels (0.7% to 1 .2% in TBE [Section 2.6.1] or TAE electrophoresis buffer [40 mM Tris-HCl, 20 mM glacial acetic acid and 2 mM Na2EDTA, pH 8.2]) by phenol freeze extraction (method based upon Thuring et al., 1975).

After gel electrophoresis to separate DNA fragments, the DNA fragment(s) of interest were excised from the gel with the minimum amount of excess agarose and placed in 1.5 ml microcentrifuge tubes. The agarose was melted at 65°C, covered with Tris- equilibrated phenol (Section 2.6.8), mixed by inversion, and the mixture was frozen (generally) overnight at -20°C. The tube was then centrifuged in a microcentrifuge for 1 0 min. The aqueous phase was recovered, extracted with

phenol/chloroform (Section 2.8) and precipitated with ethanol (Section 2.9). 2.14 DNA LIGATIONS

2.14.1 CAP-Treating of Vector DNA

Approximately 1 .0 Jlg vector DNA was digested to completion by the appropriate restriction endonuclease (Section 2. 1 1). 1.0 Jll calf alkalinephosphatase(CAP) was added and the mixture was incubated for 30 min at 37°C. An additional 1 .0 Jll CAP was added and the mixture was incubated for an additional 30 min at 37°C. The mixture was heated at 65°C for 5 min, then phenol/chloroform extracted (Section 2.8), and the precipitated DNA (Section 2.9) resuspended in MilliQ water. 2.14.2 Ligation

Ligation mixtures contained 4.0 Jll of 5 x ligation buffer (Section 2.6.9), an equimolar ratio insert:vector, (at least 20 ng DNA insert and at least 20 ng vector DNA, generally pretreated with calf alkaline phosphatase, Section 2.14.1), 1 .0 Jll of 1!10 or 1 .0 Jll of undiluted T4-DNA ligase (New England Biolabs), and MilliQ water (to 20 Jll). Ligation mixtures were left in a refrigerator ( 10- 14°C) overnight. To check that ligation had occurred, 2.0 Jll of the ligation mix was removed prior to addition of T4-DNA ligase. 2.0 Jll of SDS sample buffer (Section 2.6.4) was added and the sample was examined on an agarose gel (Section 2.12) alongside a 2.0 Jll sample (with 2.0 Jll SDS sample buffer) removed after addition of T4-DNA ligase and overnight ligation.

2 . 15 P REPARATION AND T RANSFORMATION O F C OMPETENT CELLS

An overnight, LB broth (Section 2.5. 1) culture (30°C) of E. coli strains HB 1 0 1 or MC 1022 was diluted 1/100 into 25 ml LB broth, then incubated with shaking at 37°C for 2 h (to an OD600 of approximately 0.4). Cells were harvested by centrifugation at 3000 x g _{for 10 min at 4°C in glass Corex tubes. The pellet was} carefully resuspended in 10 ml _{ice cold (4°C) 60 mM calcium chloride (suspension} was performed in the Corex tube) and stored on ice for 20 min. The cells were pelleted at 3000 x g _{for 10 min at 4 °C and carefully resuspended in 250 Jll ice cold}

( 4 °C) 60 mM calcium chloride. All procedures, except, centrifugation were performed in a cold room (approximately 4-5°C).

50 j.Ll of competent cells prepared as described above were mixed with 5 j.Ll ligated DNA (Section 2. 14.2) and 45 j.Ll TEC buffer (Section 2.6.6) in an ice cold (4°C) microcentrifuge tube and placed on ice for 1 h. The cells were then heat-shocked at 42°C for 2.5 min and placed on ice for a further 15 min. 0.9 ml _{of LB broth} (Section 2.5 . 1 ) was added and the cells were shaken within the microcentrifuge tube at 37°C for 90 min. 100 j.Ll aliquots were plated out onto appropriate selective media (Section 2.2). Plates were incubated overnight and several (generally 1 0) transformants were single colony purified. Single colony purified transformants were checked for the presence of the appropriate plasmid by DNA isolation (Section 2.7.2), digestion of the isolated DNA with (an) appropriate restriction endonuclease(s) (Section 2. 1 1) and examination of the digestion fragments for the presence of both vector and insert by agarose gel electrophoresis (Section 2.12). In addition to ampicillin (S ection 2.2) , plates u sed in the selection for transforrnants of MC1022 (used where the vector was pUC1 1 8) were spread with 20 j.Ll of a 20 mg/ml solution of X-gal (5-bromo-4-chloro-3-indolyl-J)-D­ galactoside, BRL). White (recombinant) colonies were selected for single colony purification and plasmid identification as described in the previous paragraph. 2.16 SOUTHERN BLOT TECHNIQUE

DNA to be transferred was separated by overnight electrophoresis, stained, visualised and photographed as describe in Section 2. 12 and the gel dimensions were measured after removal of the gel portion "above" the wells.

The gel was placed in a dish containing 0.25 M hydrochloric acid and gently agitated for 15 min. The hydrochloric acid solution was poured off and the gel was immersed in 0. 5 M sodium hydroxide-0.5 M sodium chloride , with gentle agitation, for 30 min. The NaOH-NaCl solution was drained and the gel was immersed in 0.5 M Tris (pH 7.4)-2.0 M sodium chloride, with gentle agitation, for

15 min. The gel was fmally washed for 2 min in 2 x SSC (Section 2.6. 1 1).

While the gel was being treated, a plastic trough with wells at each end was prepared by placing two sheets of 20 x SSC (Section 2.6. 10) soaked Whatman

3MM chromatography paper in it such that the ends of the paper projected into the wells. The wells were then filled with 20 x SSC to just below the horizontal surface of the paper between the wells. A sheet of Gladwrap was placed over the trough and pressed flat. A grid 2 _{mm less than the gel size was marked on the} Gladwrap and removed. The treated gel was placed, inverted, over the grid such that the edges of the gel overlapped the edges of the grid. A piece of nitrocellulose or nylon membrane (Hybond-C extra or Hybond-N, Amersham), cut to 2 mm greater than the gel size and presoaked in 2 x SSC, was placed over the gel ensuring that no air bubbles were present. Two pieces of Whatman 3MM chromatography paper, cut 2 mm _{less than the gel size and presoaked in} 2 _{x SSC,} were placed over the membrane. Two pieces of Whatman 3MM chromatography paper (unsoaked) were placed upon the two soaked pieces of 3MM paper. A stack of paper towels approximately 50 mm deep was placed upon the chromatography paper, followed by a flat metal or plastic tray and a weight sufficient to keep the entire stack flat.

After overnight transfer, the apparatus was disassembled and the membrane was washed for 5 min in 2 x SSC, then baked under vacuum at 80°C for 2 h.