METHODS OF DMA AND RNA MANIPULATION

2.1.1 GROWTH AND MAINTENANCE OF BACTERIAL CULTURES.

Details of the Escherichia coli (E.coli) strains used in these studies are shown in Table 2.1.1.

(i) Maintenance.

Colonies were streaked onto giucose/minimal media plates (lxM9 salts, ImM MgS04 , ImM thiamine-HCl, O.lmM CaCl2 , 0.2% w/v glucose, 1.57. Bacto-agar: 10xM9 salts is 60g Na2HP04 , 30g

lvH-/ r04 , 10a NH4C1, 5g NaCl per litre). The plates were

incubated inverted overnight, at 37C, before being sealed with parafilm. They were stored at 4C for periods of up to one month before re-plating.

(ii) Growth on solid media.

For the selection of cells transformed witn piasmia u n a, bacteria were suspended in L-Broth (1% w/v Bacto-tryptone, 0.5% w/v Bacto-yeast extract, 1% w/v NaCl) and spread evenly onto L- Amp plates (L-Broth made 1.5% w/v Bacto-agar and 100)jg/ml Ampicillin). The plates were incubated inverted overnight at 3/C. Single colonies were picked from these plates and streaked onto fresh L-Amp media. These colonies could be stored for up to two weeks at 4C.

(ill) Growth in liquid aedia.

To create an overnight culture, lOmls of L-Broth was inoculated with a single colony picked from a plate, and incubated overnight at 37C, 200rpm, in an orbital shaker. To select for bacterial colonies transformed with plasmids bearing antibiotic resistance to ampicillin, the antibiotic was added to the L-Broth before incubation to a final concentration of 100^ig/ml.

2.1.2 PREPARATION OF PLASMID DNA.

(i) Small-scale preparation of plasmid DNA - "mini-preps".

The alkaline-lysis method of Maniatis at al (1989) was used for the rapid preparation of plasmid DNA.

(ii) Large-scale preparation of plasmid DNA.

The method used was based on the method described by Holmes and Quigley (1981). An overnight culture was grown from

a colony transformed with the plasmid of interest. The

following day, 400mls of SOB medium (2% w/v Bacto-tryptone, 0.5% yeast extract, lOmM NaCl, 2.5% KC1, lOmM M g C ^ : made lOOug/ml Ampicillin) was inoculated with 4mls of this overnight culture. The 400ml culture was incubated overnight as described above. The cells were then pelleted at 5,000rpm for lOmin at 4C, and resuspended in 7.2mls SET (25mM Tris-HCl pH8.0, lOmM EDTA, 15% w/v sucrose). To this, 7.2mls lysozyme solution

(4mg/ml lysozyme in SET: made fresh) was added and the

suspension incubated at room temperature for 5min. Then 12mls of 10% w/v Triton X-100 was added and the solution boiled over a bunsen flame until it became gelatinous and stuck to the walls of the container. The container was briefly immersed in a boiling water bath, before plunging it into icy water.

The gelatinous cell mass formed after these treatments was centrifuged at 20,000rpm for 30min at 4C. The supernatant from this centrifugation was transfered to a fresh tube and 0.5 volumes 7.5M ammonium acetate added. The solution was incubated

on ice for 20min to precipitate protein, and was then

centrifuged at 15,000rpm for lOmin at 4C. The protein pellet was discarded, 0.7 volumes isopropanol added, and the nucleic

acid left to precipitate at -20C for 10 minutes. The

precipitated nucleic acid was pelleted at 15,000rpm for lOmin at 4C. The pellet was drained and the nucleic acid resuspended in 4mls Low TE (lOmM Tris-HCl pH8.0, O.lmM EDTA).

RNA, chromosomal and nlcked-plasmld DNA by resolution on a Caesium chloride gradient. 4.3g caesium chloride was dissolved in and 0.5ml ethidium bromide solution (5mg/ml in water) added to the 4ml isolated nucleic acid. The solution was sealed in a 4.5ml Beckman 'quick-seal' ultracentrifuge tube and centrifuged at 55,000rpm, 20C for 6 hours in a Beckman Vti65 rotor. The lower, supercoiled plasmid band was removed using a syringe and needle, and the ethidium bromide removed by three extractions with isopropanol which had first been equilibrated with caesium chloride-saturated TE (lOmM Tris-HCl pH8.0, ImM EDTA). Four volumes of Low TE was then added to the aqueous phase, the solution made 0.3M with respect to sodium acetate, and the DNA

precipitated by adding 2 volumes of absolute ethanol and

incubating at -20C for 2 hours to overnight.

Finally, the DNA was pelleted at 15,000rpm for lOmin at 4C, the pellet washed in 70% ethanol, dried in vacuo, and resuspended in 400pl water.

(iii) Single-stranded (ss) N13 preparations.

The single-stranded (+ strand) form of vectors and constructs derived from the bacteriophage M13 (Messing, 1986) was prepared according the method of Schreier and Cortese (1979).

(iv) Replicative fora (RF) M13 preparations.

2ml of 2xTY/magnesium (1.6% w/v Bacto-tryptone, 1% w/v yeast extract, 0.5% w/v NaCl, made lOmM MgC^)» was inoculated with an M13 plaque taken from a fresh plate - using a glass pipette to remove the plaque in the form of an agar plug. 0 . 0 1

volumes of an overnight culture of E.coli TGI cells was added, and the culture incubated in orbital shaker for 5-6 hours at 37C and 300rpm. The cells were then pelleted in a microfuge for

5min and the supernatant, containing infectious ssM13

particles, was stored overnight at 4C.

400ml of 2xTY/magnesium was inoculated with 0.01 volumes of an overnight culture of E.coli TGI cells and incubated at

37C, 200rpra, until the cell culture was in mid-log phase (taken to be when the optical density of the culture at 600nm reached

approximately 0.5). The suspension of infectious ssM13

particles was then added, and the culture grown for a further 4

hours at 37C, 300rpm. The RF form was then prepared as

described for large-scale plasmid preparations above.

2.1.3 STORAGE OF DNA.

All preparations of purified plasmid and M13 (ss and RF) DNA were stored frozen in H2O or TE at -20C.

2.1.4 QUANTITATIVE ESTIMATION OF DNA.

(i) Spectrophotometric estimation.

The concentration of DNA in an aqueous solution was estimated by measuring the absorbance at 260nm (&260^ an<* converting this value to mg/ml, viz; 1 OD unit is equivalent to 40ug/ml for double-stranded (ds) DNA or 30ug/ml for ssDNA. As an indication of purity, the level of protein contamination in a DNA solution was monitored by determination of the A26o:A280 ratio. A solution of pure nucleic acid gives an A260:A280 ratio of 1.8 (Maniatis et al, 1989).

(ii) Gel analysis.

To quantitate fragments of plasmid DNA, a known aliquot was run on an agarose gel (as described below) alongside 500ng of bacteriophage lambda DNA that had been previously digested with Hindlll and EcoRI and heated at 65C for 5min to dissociate the cos sites. The bands were visualised by placing the gel on a UV transilluminator, and the intensity of the fragment compared to those of the lambda bands. The concentration of DNA was then determined by reference to Table 2.1.4.

TABLE 2.1.4

SIZES AND COHCEHTRATIOHS OF LAMBDA FRAGMENTS AFTER