Generation of Recombinant DNA Molecules

2.2.1.1 Digestion of DNA using restriction endonuclease enzymes

Restriction endonucleases cleave at specific sites on the DNA molecule. The enzymes were diluted to a final concentration o f 1 unit/pl and used with the manufacturer’s recommended buffer. The digest was incubated at the appropriate temperature (37®C for most enzymes) for 2-3 hours. Some enzymes also required the addition o f BSA and/or Triton. The sample could then be used in agarose gel electrophoresis which separates differently sized DNA molecules.

2.2.1.2 Agarose Gel Electrophoresis

This technique is used to resolve and visualise differently sized DNA molecules. Agarose acts as a molecular sieve and smaller DNA molecules travel through agarose faster than larger DNA molecules. A solution of agarose ranging from 0.5-2% (w/v) was prepared by boiling powdered agarose in electrophoresis buffer and allowed to cool to SO^C. The percentage o f agarose in solution varied depending on the size of the DNA molecule (generally, for DNA molecules in the size range o f 200 bp-1 kb, a 2% agarose gel was prepared. For molecules in the range o f 10-20 kb a 0.5% agarose gel was used). The gel was then poured into a preassembled casting tray with the appropriate comb in place to form wells. Thinner combs tended to give better resolution. Once set, the gel was placed in the electrophoresis apparatus (Pharmacia GNA-200, BRL Horizon 11.14 and 58) and covered with electrophoresis buffer. DNA has an overall negative charge and migrates towards the positive anode. The migration of the DNA molecules is inversely proportional to the logjo o f the molecule’s size. In addition markers o f known DNA size were electrophoresed on the same gel at the

same time. The loading buffer contains Ficoll-400 which prevents the sample from floating out o f the well (glycerol can also be used) and bromophenol which is a dye that migrates at approximately 500 bp in a 1% gel and can be used to estimate the distance the DNA has migrated through the gel. The gel was then stained in a 0.1 mg/ml ethidium bromide solution for 30 minutes and then visualised on an ultraviolet transilluminator at a wavelength o f 302 nm. Gels were photographed using a Polaroid Land camera on Kodak technical pan 4445 film and processed through an AGFA Curix 60 developer.

2.2.1.3 Extraction of DNA from Agarose Gels

The DNA band o f interest was excised from the agarose using a clean sharp blade and purified using the Qiaex II Extraction kit (Qiagen). The principle involves melting the agarose at 50°C in the presence of a homogenous suspension of small particles that bind to the DNA once it has been released from the agarose. The particles are then washed to remove any residual agarose before the DNA is eluted from the beads using TE buffer pH 8. The procedure was carried out according to the manufacturer’s instructions.

2.2.1.4 Alkaline Phosphatase Treatment of Digested DNA

The treatment o f restriction endonuclease-digested DNA with calf intestinal alkaline phosphatase (Boehringer Mannheim) prevents compatible ends of cut DNA rejoining during ligation reactions by dephosphorylating the 5' end o f the molecule. One unit of alkaline phosphatase was added to the DNA in the last half hour o f a restriction endonuclease digestion of DNA. The phosphatase enzyme was subsequently removed by electrophoresis of the molecule. Dephosphorylation was not necessary if the restriction enzymes used yielded incompatible ends.

2.2.1.5 Phenol/Chloroform Extraction

A solution containing 50% phenol, 48% chloroform and 2% isoamylalcohol was used to remove any contaminants (such as salts and proteins) in a DNA preparation. An equal volume o f this solution was added to the DNA solution. After vigorous shaking on a vortex, the sample was centrifuged at 13000rpm for 3 minutes. The DNA solution was removed from the phenol and the chloroform using a pipette.

2.2.1.6 Ethanol Precipitation of Nucleic Acids

DNA (or RNA) can be purified from solution by precipitating the nucleic acid in 0.7 volumes of isopropanol at room temperature. The sample was then centrifuged at 13000rpm for 30 minutes. The resulting pellet was washed in 70% ethanol, allowed to air dry and then allowed to dissolve in either water or TE pH 8. DNA tends to dissolve quicker in TE than H2O as it is more alkaline.

2.2.1.7 Ligation of DNA Fragments

DNA fragments with either cohesive compatible ends or blunt ends can be ligated together to produce a circular molecule. One unit o f T4 DNA ligase was used in a 20 fil reaction volume with the manufacturer’s recommended buffer containing the appropriate concentration of rATP and/or Mg2+. For vector-insert ligations the DNA insert and vector were added in a 3:1 ratio if the ends were cohesive (either 3’ or 5’ overhangs) or a 10:1 ratio if the DNA molecules were blunt-ended. The reaction was incubated at 17®C overnight (or 4 hours at 37°C) before being diluted to a final volume of 100 pi in dH2 0. 20 pi was then use to transform competent cells.

2.2.1.8 Preparation of Competent Cells (Chung & Miller, 1988)

The use o f competent cells increases the bacterial host's ability to take up exogenous DNA and reduces the difficulties in obtaining recombinant clones. Bacterial cells were grown to early log phase (OD^qo ^ 0 4) in L-broth and centrifuged and resuspended in 1/10 of the volume in prechilled transformation and storage buffer (L- broth pH 6.1 containing 10% polyethylene glycol (MW=3,350), 20 mM Mg^^ (10 mM MgClj + 1 0 mM MgSOJ, and 5% DMSO) at 4°C. After incubating on ice for 20 minutes, the cells were aliquoted and frozen in dry ice, then stored at -70°C. PEG induces competence whilst DMSO acts as a cryoprotectant. thus providing readily available frozen stocks o f competent cells.

2.2.1.9 Use of Competent cells in Bacterial Transformations

Competent cells were thawed slowly on ice as their level of competence is temperature sensitive. 20 pi of the diluted ligation reaction (or 0.5 pg uncut plasmid) were added to 100 pi of cells in a precooled microfuge tube and mixed by gentle pipetting. The tube was returned to ice for a further 30 minutes. 700 pi L-broth was then added and the tube left at 37°C for 1 hour. 150 pi o f the cells were plated out onto L-agar containing the appropriate selection antibiotic. The addition o f 35 pi of 50 mg/ml X-gal and 20 pi of 100 mM IPTG per L-agar antibiotic plate was optional if blue/white screening of recombinant clones was required.