PRINCIPIOS Y DERECHOS CONSTITUCIONALES DE LA PRISIÓN

Bacterial restriction endonucleases were first discovered in 1969. In vivo, these enzymes are involved in recognising and cutting foreign DNA entering the cell and their most likely role is protecting the bacteria against phage infection. This property o f recognising specific DNA sequences has been very useful as a molecular cloning technique (Ausubel et al. 1993). The restriction enzymes most commonly used in genetic manipulation are the Type II class. These recognise sequences o f four (4- cutters) or six (6-cutters) bases and cleave double-stranded DNA within the recognition sequence, and at its complement, into fragments which can be separated by gel electrophoresis. Some enzymes generate blunt eruied fragments as they cut in the same position on each strand and others yield fragments with staggered (sticky) ends.

DNA samples from the 'mini-prep' procedure were diluted to a working concentration o f 100 ng DNA/pl m i x TE and retained on ice until required.

For each dUuted sample the following four 10 pi restriction digest reactions were set up:

Tube

sample (pi) buffer (10x;pl) sterile water (pi) EcoRI (pi) Xhol (pi) Tube 1 2 3 4 Description

uncut plasmid control single digest with EcoRl single digest with Xhol

The reactions were flick-mixed and then incubated at 37°C for 1-2 h.

2.2.2.3 Agarose gel electrophoresis

Gel electrophoresis allows separation o f DNA fragments on the basis o f size. DNA molecules have a net negative charge at neutral p H so when placed in an electric fie ld they migrate towards the positive terminal. The separation matrix may either be an agarose or a non-denaturing polyacrylamide gel depending on the size and amount o f DNA ^o be analysed and on the resolution required. Agarose forms a gel by hydrogen bonding when in cool aqueous solution, and the pore size depends on agarose concentration. Larger pores, effective for separation o f molecules from 0.2 kilobases (kb), are obtained using 1-1.5% agarose; fo r molecules up to 25 kb, 0.3- 0.5% agarose is required. In experiments described here, 0.8% agarose was routinely used to cover the size range o f 0.5-10 kb. The migration speed o f DNA o f 0.1-20 kb is determined almost entirely by length and is independent o f base composition. The gel pores act as a sieve thus smaller fragments have the highest mobilities. Molecules o f the same size migrate through the gel as a band which gradually increases in width due to diffusion.

The DNA is detected by ethidium bromide (EtBr) staining. This dye is incorporated into the gel and intercalates between adjacent pairs o f bases emitting red fluorescence on exposure to ultraviolet (UV) light (260-360 nM). The limit o f detection is 5-10 ng DNA. The size o f the molecules in any band can be deducedfrom standard molecular weight markers run in a parallel lane on the gel.

Agarose (0.8%; Gibco-BRL Life Technologies Ltd, Paisley, Scotland, U.K.) in 50 ml 1 X TBE gel running buffer (Appendix A) was melted in a 650W microwave oven (2 min on low power setting) removed, stirred and returned to boil. The agarose was allowed to cool and 5 pi EtBr (5 mg/ml) added. The agarose/EtBr was poured into a minigel electrophoresis tray (Gel electrophoresis apparatus GNA-100, Pharmacia Biotech Ltd, Milton Keynes, Bedfordshire, U.K.) containing a plastic comb to form the wells. The gel was allowed to set for 15-30 min and mounted into the gel tank.

Gel running buffer was poured over the gel until the tank was fWl and all the wells were completely submerged.

The 10 pi digests, from section 2.2.2.2, were prepared for loading by the addition of 2 pi gel loading buffer (Stop solution; Appendix A) , mixed thoroughly and pulsed in a microfuge. A suitable size marker was prepared from a Hind HI restriction digest of lambda (^) DNA (fragment size range: 125 - 23130 base pairs; NBL Gene Sciences Ltd., Cramlington, Northumbria, U.K.; Appendix A). Size marker (1 pg) was prepared by the addition o f 3.3 pi stock (300 pg/ml) to 6.7 pi sterile water. This maintained a total volume of 10 pi, equivalent to the total volume o f each digest. An approximate concentration o f DNA in any restricted fragment was assigned based on a known conversion o f the size marker bands assuming a loading o f 1 pg (Appendix A).

The samples and size marker (12 pi) were loaded into adjacent wells using a fine Gilson pipette and the gel run at 50 mV (approximately 35 mA, depending on gel running buffer), for 1 . 5 - 2 hours. The gel was run slowly and for a relatively long time to improve separation o f the expected insert and bluescript SK+ plasmid vector.

The gel was viewed under an ultraviolet transilluminator (UVP Ultraviolet Transilluminator, Genetic Research Instruments (GRI), Gene House, Dunmow, Essex, U.K.) and photographed using Polaroid Tipo 667 black and white instant pack film (Fabrique au Royaume-Uni par Polaroid (U.K.) Ltd., St Albans, Hertfordshire, U.K.).

2.2.2.4 Large plasmid preparation: PEG method

The large scale polyethylene glycol (PEG) method o f plasmid DNA preparation described was based on that previously reported by Sambrook et al. (1989).

A positive glycerol stock of the largest OTR cDNA clone was used to inoculate 1 ml L-broth containing 1 pi ampiciUin (50 mg/ml) in 12.5 ml sterile polystyrene tubes

(Falcon). The tubes were shaken in a 37“C vertical rotary incubator (CM 100 cell mixer, Luckham Ltd, Burgess-Hill, Sussex, U.K.) for 6 hours, or until the cultures had reached log phase o f growth. Log phase culture (0.5 ml) was added to 500 ml (1:1000 dilution) L-broth containing 0.5 ml ampiciUin (50 mg/ml) in a sterile, baffled glass flask (PhiUip Harris Scientific, London, U.K.). The flask was placed in an orbital shaker (GaUenkamp) at 37“C, 300 cycles/minute, and the culture grown overnight to stationary phase.

The culture was transferred to two 250 ml rotor buckets (Beckman Instruments (U.K.) Ltd) and centrifuged in a Beckman 60' J2-21 centrifuge at 2500g (rotor JA-14) for 20 min at 4°C. The supernatant was poured to waste and the peUets resuspended in 4 ml 50 mM Tris/HCl (pH 8.0) containing 25% (w/v) sucrose (Appendix A). The resuspended peUets were transferred to 50 ml Oakridge tubes (Phillip Harris) and the final volume o f each tube recorded. Lysozyme was added to a final concentration of 1 mg/ml and mixed by gently rolling the tubes. The tubes were left on ice for 15 m in to aUow complete ceU lysis. EDTA was added to final concentration o f 10 mM, the tubes left on ice for a fiuther 15 m in and 0.5 volume 3 x Triton buffer (Appendix A) added to break down the ceU membranes. The Triton was mixed in gently, but thoroughly, and the tubes left on ice for 30 m in before being centrfuged at 25500g (rotor JA-20) for 60 min at 4°C. The supernatants were careftdly removed, transferred to sterile 50 ml conical Falcon tubes and made up to 0.5M with respect to NaCl (the volume made up to 10 ml with 50 mM Tris/HCl (pH 8.0) and then 1 ml 5M NaCl added). The supernatants were extracted twice with an equal volume o f equilibrated phenol:chloroform (pH 8.0; Appendix A). The second aqueous phase was extracted with an equal volume o f chloroform:isoamyl alcohol (24:1), saturated with water, and centrifuged as above (2500g, 10 min). The final aqueous phases were transferred to fiesh tubes and 10% (w/v) polyethylene glycol (PEG, mwt 8000; Sigma) added. The tubes were placed in a 37°C water bath, shaken at regular intervals, to allow all o f the PEG to dissolve, and placed at 4°C overnight to enable the PEG to precipitate out the plasmid DNA.

and centrifuged at 14500g (rotor JA-20.1) for 20 min at 4°C. The supernatants were poured to waste and the pellets dissolved in 500 pi O.IM Tris/HCl (pH 8.0) and transferred to sterile Eppendorf tubes. Ten pi pre-boüed RNAse (10 mg/ml) was added, mixed by gentle pipetting and incubated at 37°C for 45 min. An equal volume of PEG buffer (Appendix A) was added and the tubes incubated on ice for 60 min. The tubes were then centrifuged at 11600g for 10 min, the supernatants poured to waste and the pellets redissolved in 0.5M NaCl/10 mM Tris/HCl (pH 8.0). The resuspended peUets were extracted twice with phenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform:isoamyl alcohol (24:1) as described in the 'mini- prep' procedure (section 2.2.2.1). To the final aqueous phase was added 0.1 volume 4M NaCl and 2 volumes 95% ethanol (-20°C) and the tubes placed at -70®C for 20 min or -20“C for 2 hours.

The ethanol precipitates were centrifuged (11600g, 10 min) and the peUets washed with 70% ethanol (-20“C; ethanol precipitation). AU the ethanol was removed and the peUets vacuum dried in a Speedvac™ dryer (Salvant Instruments Inc., Farmingdale, New York, U.S.A.) before being taken up in 100 pi 1 x TE. The ODjgo o f the resulting plasmid DNA suspensions was then measured on a mass spectrophotometer.