EL PROBLEMA SOCIAL Y SU RELACIÓN CON LAS DINÁMICAS

The following E. coli strain was used for plasmid preparation:

E. coli strain MC1022 araD139 del(ara leu)7697 <!>80dlacZ MI5 galU galK strA (Casadaban & Cohen, 1980).

2. 1 3 . 1 pUC1 9 cloning.

The buffers and reagents used in pUC19 cloning are detailed in Appendix 2.5.

C omplementary DNA was cloned into the bacterial plasmid pUC 1 9 and transformed into E. coli strain MC1 022. The plasmid pUC19 has a polylinker region, which when cloned into interrupts the a-peptide of the B-galactosidase gene, resulting in loss of the ability to metabolise 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (BCIG). When grown on agar containing BCIG, recombinant plasmid colonies appear colourless compared with · non-recombinant plasmids with an intact B-galactosidase gene which appear blue (lac+). Plasmid pUC 19 also contains a _�-lactamase gene conferring on the transformed bacteria the ability to metabolise ampicillin. Thus white colonies growing on media containing ampicillin usually represent colonies containing a cDNA insert in the genome of plasmid pUC 1 9. cDNA was usually cloned into the Smal polylinker site of pUC19.

2. 1 3. 1 . 1 Ligation of cDNA to pUC19.

Ligation of cDNA to plasmid pUC19 was accomplished by adding 1 ).11 of the cDNA produced in the preceding reaction (2. 13) to 100 ng Smal cut pUC 19 in ligase buffer (25 m M Tris.HCl (pH 7.6), 5 mM MgC12, 0.5 mM ATP, 0.5 mM DTT, 2.5% (w/v)

polyethylene glycol-8000; BRL) and 0. 1-0.4 units T4 DNA ligase (BRL). The ligation was allowed to proceed 2- 12 h at room temperature.

Ligation control reactions of vector only, vector and ligase, and competent cells only were carried out as well as the ligation reaction to assess the extent of background deletion ligations and contamination.

2. 13. 1 .2 Transformation of E. coli strain MC 1022.

Competent cells of E. coli strain MC1022 were produced by inoculating 1 0 ml of 2x TY (bacto-tryptone 1 6g/l, bacto-yeast extract 10g/l, NaCl 5gll) with a loopful of a stock culture of MC1 022. Following overnight incubation at 37 C with agitation at 225 rpm,

100 ml of 2x TY was inoculated with 1 ml of the overnight culture. This was grown for 1 -2 h at 37 C with shaking until mid log phase (OD 660 nm of 0.4-0.7) after which the culture was placed on ice for 1 h. The E. coli cells were collected by centrifugation at 2000 rpm for 1 0 min. in the coldroom and resuspended in 1 0 ml ice-cold 50 mM CaC12• After 30 min. on ice, the cells were again centrifuged at 2000 rpm for 5 min. and resuspended in 4 ml ice-cold 50 mM CaC12 for 30 min. or until required.

Five 111 of ligation reaction was added to 20 Ill water in a microfuge tube, and chilled on ice for 5 min. Competent cells, 50 fll per tube, were added to chilled and diluted (5 111 ligation reaction in 20 fll water) ligated pUC 1 9 and the mixture chilled on ice for 20 min. or longer. Cells were heat shocked for 1 min. at 42 C and then placed on ice for 1 min. Transformed cells were transferred to disposable test-tubes containing 0.5 ml 2x TY and incubated at 37 C for 1 h with shaking (225 rpm). Dividing cells were plated on two L

plates containing 100 !lglml ampicillin and 40 !lglml BCIG per treatment and incubated at 37 C overnight.

2. 13. 1 . 3 Small-scale Plasmid DNA Isolation from E. coli (Minipreps).

White colonies were picked off L plates containing 100 11g/ml ampicillin and 40 !lglml BCIG with a sterile toothpick, concurrently subcultured and inoculated to 3 ml of 2x TY containing 100 11g/ml ampicillin. Liquid cultures were incubated on a shaker at 225 rpm at 37 C overnight. Following incubation, the inoculated broth was aliquoted into

microfuge tubes and centrifuged at 6500 rpm for 2 min. The supernatant was removed by aspiration and the pellet resuspended in 200 !11 STET (Appendix 2.5) buffer. Bacterial cell membranes were lysed by the addition of 5 !ll lysozyme (40 mg/ml) and contaminating RNA removed by the addition of 1 111 RNase A (10 mg/ml) to the resuspended pellet and incubation at room temperature for 5 min. The mixture was boiled for 45 sec., then centrifuged at 13 000 rpm for 45 min. in a Heraeus microfuge to pellet cell debris and chromosomal DNA. The pellet was removed and discarded with a sterile toothpick, and 20 !11 of the supernatant was added to 2 111 of l Ox BPB (0.25% bromophenol blue (w/v), 25% Ficoll type 400 (w/v)) and electrophoresed (Chpt. 2. 10.5). If inserts were larger than 400 bp, the remaining supernatant was further purified by precipitation of the DNA with 1 0 111 CTAB (cetyltrimethylammonium bromide; Del Sal et al., 1 988). The precipitate was collected following centrifugation at 13 000 rpm for 5 min. and the supernatant discarded. The pellet was resuspended in 300 !11 1.2 M NaCl, to which was added 750 111 absolute

ethanol. Following incubation at - 1 8 C for 1 h or longer, the precipitated DNA was pelleted (centrifugation at 13 000 rpm for 15 min), washed with 70% ethanol, vacuum dried and resuspended in sterile DDW.

2. 13.1.4 Large-Scale Plasmid DNA Isolation From E. coli (Maxipreps)

White colonies which had an insert of interest were prepared for dideoxy sequencing using an alkaline denaturation method which results in larger quantities of DNA of greater purity than miniprep DNA (Anonymous, 1 989). Following denaturation, selective renaturation of plasmid DNA was achieved by neutralisation of the solution. Precipitation using PEG (polyethylene glycol) was included to remove contaminants which could interfere with restriction digests or sequencing procedures.

Terrific broth (Appendix 2.5) containing ampicillin (100 jlg/ml) was inoculated with a loopful of bacteria from colonies of interest and incubated with shaking (225 rpm)

overnight at 37 C. Cells were collected following centrifugation at 5000 g for 1 5 min. at 4 C and the pellet was resuspended in 6 ml fresh lysis buffer (25 mM Tris.HCl (pH 7 .5), 10 mM EDTA, 15% sucrose, 2 mg/ml lysozyme). To the resuspended pellet, 12 ml of 0.2 M NaOH and 0. 1 % SDS was mixed by inversion and incubated on ice for 10 min.

Sodium acetate (7 .5 ml, 3 M, pH 4.6) was added and mixed by inversion and lysis completed following incubation on ice for 10 min. The lysed cells were removed by centrifugation for 10 min. at 13 000 rpm. To the supernatant, 500 jlg of RNase A was

added and incubated at 37 C for 2 h, after which the solution was extracted twice with phenol and chloroform and once with chloroform followed by precipitation with ethanol. DNA was pelleted by centrifugation (13 000 rpm for 20 min.) and dissolved in 1 .6 ml sterile DDW. To the resuspended pellet was added 0.32 ml 5M NaCl and 2 ml 13% PEG-8000 and the suspension was incubated at 4 C overnight. The purified DNA was pelleted by centrifugation at 10 000 rpm for 10 min., washed with 70% ethanol, vacuum dried and resuspended in sterile DDW.